WO2013098562A2

WO2013098562A2 - Enzyme method

Info

Publication number: WO2013098562A2
Application number: PCT/GB2012/053274
Authority: WO
Inventors: Ruth Moysey; Andrew John Heron; Szabolcs SOEROES
Original assignee: Oxford Nanopore Technologies PLC
Current assignee: Oxford Nanopore Technologies PLC
Priority date: 2011-12-29
Filing date: 2012-12-28
Publication date: 2013-07-04
Anticipated expiration: 2014-06-29
Also published as: BR112014016112B8; CA2861808C; EP2798084A2; BR112014016112A2; CN104126018B; JP2015503917A; AU2012360244A1; CN104126018A; CA2861808A1; AU2012360244B2; EP2798084B1; KR102086182B1; WO2013098562A3; KR20140108706A; US10385382B2; JP6228128B2; US20140335512A1; BR112014016112B1

Abstract

The invention relates to a new method of characterising a target polynucleotide. The method uses a pore and a RecD helicase. The helicase controls the movement of the target polynucleotide through the pore.

Description

ENZYME METHOD

Field of the invention

Background of the invention

There is currently a need for rapid and cheap polynucleotide (e.g. DNA or RNA) sequencing and identification technologies across a wide range of applications. Existing technologies are slow and expensive mainly because they rely on amplification techniques to produce large volumes of polynucleotide and require a high quantity of specialist fluorescent chemicals for signal detection.

Transmembrane pores (nanopores) have great potential as direct, electrical biosensors for polymers and a variety of small molecules. In particular, recent focus has been given to nanopores as a potential DNA sequencing technology.

When a potential is applied across a nanopore, there is a change in the current flow when an analyte, such as a nucleotide, resides transiently in the barrel for a certain period of time. Nanopore detection of the nucleotide gives a current change of known signature and duration. In the "Strand Sequencing" method, a single polynucleotide strand is passed through the pore and the identity of the nucleotides are derived. Strand Sequencing can involve the use of a nucleotide handling protein to control the movement of the polynucleotide through the pore.

Summary of the invention

The inventors have demonstrated that a RecD helicase can control the movement of a polynucleotide through a pore especially when a potential, such as a voltage, is applied. The helicase is capable of moving a target polynucleotide in a controlled and stepwise fashion against or with the field resulting from the applied voltage. Surprisingly, the helicase is capable of functioning at a high salt concentration which is advantageous for characterising the

polynucleotide and, in particular, for determining its sequence using Strand Sequencing. This is discussed in more detail below.

Accordingly, the invention provides a method of characterising a target polynucleotide, comprising: (a) contacting the target polynucleotide with a transmembrane pore and a RecD helicase such that the target polynucleotide moves through the pore and the RecD helicase controls the movement of the target polynucleotide through the pore; and

(b) taking one or more measurements as the polynucleotide moves with respect to the pore wherein the measurements are indicative of one or more characteristics of the target polynucleotide and thereby characterising the target polynucleotide.

The invention also provides:

a method of forming a sensor for characterising a target polynucleotide, comprising forming a complex between a pore and a RecD helicase and thereby forming a sensor for characterising the target polynucleotide;

use of a RecD helicase to control the movement of a target polynucleotide through a pore;

a kit for characterising a target polynucleotide comprising (a) a pore and (b) a RecD helicase; and

- an analysis apparatus for characterising target polynucleotides in a sample, comprising a plurality of pores and a plurality of a RecD helicase;

a method of characterising a target polynucleotide, comprising:

(a) contacting the target polynucleotide with a RecD helicase such that the RecD helicase controls the movement of the target polynucleotide; and

(b) taking one or more measurements as the RecD helicase controls the movement of the polynucleotide wherein the measurements are indicative of one or more characteristics of the target polynucleotide and thereby characterising the target polynucleotide;

use of a RecD helicase to control the movement of a target polynucleotide during characterisation of the polynucleotide;

- use of a RecD helicase to control the movement of a target polynucleotide during sequencing of part or all of the polynucleotide;

an analysis apparatus for characterising target polynucleotides in a sample, characterised in that it comprises a RecD helicase; and

a kit for characterising a target polynucleotide comprising (a) an analysis apparatus for characterising target polynucleotides and (b) a RecD helicase.

Description of the Figures

Fig. 1. A) Example schematic of use of a helicase to control DNA movement through a nanopore. A ssDNA substrate with an annealed primer containing a cholesterol-tag is added to the cis side of the bilayer. The cholesterol tag binds to the bilayer, enriching the substrate at the bilayer surface. Helicase added to the cis compartment binds to the DNA. In the presence of divalent metal ions and NTP substrate, the helicase moves along the DNA. Under an applied voltage, the DNA substrate is captured by the nanopore. The DNA is pulled through the pore under the force of the applied potential until a helicase, bound to the DNA, contacts the top of the pore, preventing further uncontrolled DNA translocation. After this the helicase proceeds to move the DNA through the nanopore in a controlled fashion.

The schematic shows two possible methods of introducing the DNA to the nanopore: in one mode (top section) the helicase moves the captured DNA into the nanopore in the direction of the applied field, and in the other mode (lower section) the helicase pulls the captured DNA out of the nanopore against the direction of the applied field. When moved with the applied field the DNA is moved to the trans side of the membrane. In both upper and lower sections the arrows on the trans side indicate the direction of motion of the DNA and the arrows on the cis side indicate direction of motion of the helicase with respect to the DNA. When moved against the field, the DNA is moved back to the cis side of the membrane,and the DNA may translocate completely to the cis side if the helicase does not dissociate. Through substrate design, such as use of suitable leaders, one or both methods can be used at a time. The RecD family of helicases move in the 5 '-3 ' direction along the DNA. Therefore, moving the DNA with the field requires 5' down capture of the DNA, and moving the DNA against the field requires 3 ' down DNA capture. B) The DNA substrate design used in the Example.

Fig. 2. Helicase is able to move DNA through a nanopore in a controlled fashion, producing stepwise changes in current as the DNA moves through the nanopore (MspA-(B2)8). Example helicase-DNA events (140 mV, 400 mM NaCl, 10 mM Hepes, pH 8.0, 0.60 nM 400 mer DNA (SEQ ID NO: 172, 173 and 174), 100 nM Tral Eco (SEQ ID NO: 61), 1 mM DTT, 1 mM ATP, 1 mM MgCl₂). Top) Section of current vs. time acquisition of Tral 400mer DNA events. The open-pore current is -100 pA. DNA is captured by the nanopore under the force of the applied potential (+140 mV). DNA with enzyme attached results in a long block (at -25 pA in this condition) that shows stepwise changes in current as the enzyme moves the DNA through the pore. Middle) An enlargement of one of the helicase-controlled DNA events, showing DNA- enzyme capture, and stepwise current changes as the DNA is pulled through the pore. Bottom) Further enlargement of the stepwise changes in current as DNA is moved through the nanopore.

Fig. 3. Further examples of Tral Eco (SEQ ID 61) helicase controlled 400mer DNA (400mer DNA SEQ ID NOs: 172, 173 and 174) movement events through an MspA-B2(8) nanopore. Bottom) An enlargement of a section of the event showing the stepwise changes in current from the different sections of DNA as the strand moves through the nanopore. Fig. 4. Fluorescence assay for testing enzyme activity. A) A custom fluorescent substrate was used to assay the ability of the helicase to displace hybridised dsDNA. 1) The fluorescent substrate strand (50 nM final) has a 5' ssDNA overhang, and a 40 base section of hybridised dsDNA. The major upper strand has a carboxyfluorescein base at the 3 ' end, and the hybridised complement has a black-hole quencher (BHQ-1) base at the 5' end. When hybridised the fluorescence from the fluorescein is quenched by the local BHQ-1, and the substrate is essentially non-fluorescent. 1 μΜ of a capture strand that is complementary to the shorter strand of the fluorescent substrate is included in the assay. 2) In the presence of ATP (1 mM) and MgCl₂ (10 mM), helicase (100 nM) added to the substrate binds to the 5' tail of the fluorescent substrate, moves along the major strand, and displaces the complementary strand as shown. 3) Once the complementary strand with BHQ-1 is fully displaced the fluorescein on the major strand fluoresces. 4) Excess of capture strand preferentially anneals to the complementary DNA to prevent re-annealing of initial substrate and loss of fluorescence. B) Graph of the initial rate of RecD helicase activity in buffer solutions (RecD Nth and Dth SEQ IDs 28 and 35, 100 mM Hepes pH 8.0, 1 mM ATP, 10 mM MgCl₂, 50 nM fluorescent substrate DNA, 1 μΜ capture DNA) containing different concentrations of KC1 from 100 mM to 1 M.

Fig. 5. Examples of helicase controlled DNA events using a different Tral helicase, TrwC Cba (+140 mV, 10 mM Hepes, pH 8.0, 0.6 nM, 400mer DNA SEQ ID NOs: 172, 172 and 173, 100 nM TrwC Cba SEQ ID 65, 1 mM DTT, 1 mM ATP, 1 mM MgCl₂). Top) Section of current vs. time acquisition of TrwC Cba 400mer DNA events. The open-pore current is -100 pA. DNA is captured by the nanopore under the force of the applied potential (+140 mV). DNA with enzyme attached results in a long block (at -25 pA in this condition) that shows stepwise changes in current as the enzyme moves the DNA through the pore. Bottom) The bottom traces show enlarged sections of the DNA events, showing the stepwise sequence dependent current changes as the DNA is pulled through the pore.

Fig. 6. Example of current trace showing helicase controlled DNA movement using a TrwC (Atr) (SEQ ID NO: 144) helicase.

Fig. 7. Example of current trace showing helicase controlled DNA movement using a TrwC (Sal) (SEQ ID NO: 140) helicase.

Fig. 8. Example of current trace showing helicase controlled DNA movement using a

TrwC (Ccr) (SEQ ID NO: 136) helicase.

Fig. 9. Example of current trace showing helicase controlled DNA movement using a TrwC (Eco) (SEQ ID NO: 74) helicase

Fig. 10. Example of current trace showing helicase controlled DNA movement using a TrwC (Oma) (SEQ ID NO: 106) helicase. Fig. 11. Example of current trace showing helicase controlled DNA movement using a TrwC (Afe) (SEQ ID NO: 86) helicase. The lower trace shows an expanded region of the helicase controlled DNA movement.

Fig. 12. Example of current trace showing helicase controlled DNA movement using a TrwC (Mph) (SEQ ID NO: 94) helicase. The lower trace shows an expanded region of the helicase controlled DNA movement.

Description of the Sequence Listing

SEQ ID NO: 1 shows the codon optimised polynucleotide sequence encoding the MS-B1 mutant MspA monomer. This mutant lacks the signal sequence and includes the following mutations: D90N, D91N, D93N, D118R, D134R and E139K.

SEQ ID NO: 2 shows the amino acid sequence of the mature form of the MS-B 1 mutant of the MspA monomer. This mutant lacks the signal sequence and includes the following mutations: D90N, D91N, D93N, D118R, D134R and E139K.

SEQ ID NO: 3 shows the polynucleotide sequence encoding one subunit of a-hemolysin-

El l lN K147N ( -HL-NN; Stoddart et al, PNAS, 2009; 106(19): 7702-7707).

SEQ ID NO: 4 shows the amino acid sequence of one subunit of a-HL-NN.

SEQ ID NOs: 5 to 7 shows the amino acid sequences of MspB, C and D.

SEQ ID NO: 8 shows the sequence of the RecD-like motif I.

SEQ ID NOs: 9, 10 and 11 show the sequences of the extended RecD-like motif I.

SEQ ID NO: 12 shows the sequence of the RecD motif I.

SEQ ID NOs: 13, 14 and 15 show the sequences of the extended RecD motif I.

SEQ ID NO: 16 shows the sequence of the RecD-like motif V.

SEQ ID NO: 17 shows the sequence of the RecD motif V.

SEQ ID NOs: 18 to 45 show the amino acid sequences of the RecD helicases in Table 5.

SEQ ID NOs: 46 to 53 show the sequences of the MobF motif III.

SEQ ID NOs: 54 to 60 show the sequences of the MobQ motif III.

SEQ ID NOs: 61 to 171 show the amino acid sequences of the Tral helicase and Tral subgroup helicases shown in Table 7.

SEQ ID NOs: 172 to 182 show the sequences used in the Examples.

Detailed description of the invention

It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

In addition as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a pore" includes two or more such pores, reference to "a helicase" includes two or more such helicases, reference to "a polynucleotide" includes two or more such polynucleotides, and the like.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

Methods of the invention

The invention provides a method of characterising a target polynucleotide. The method comprises contacting the target polynucleotide with a transmembrane pore and a RecD helicase such that the target polynucleotide moves moves through the pore and the RecD helicase controls the movement of the target polynucleotide through the pore. One or more

characteristics of the target polynucleotide are then measured as the polynucleotide moves with respect to the pore using standard methods known in the art. One or more characteristics of the target polynucleotide are preferably measured as the polynucleotide moves through the pore.

Steps (a) and (b) are preferably carried out with a potential applied across the pore. As discussed in more detail below, the applied potential typically results in the formation of a complex between the pore and the helicase. The applied potential may be a voltage potential.

Alternatively, the applied potential may be a chemical potential. An example of this is using a salt gradient across an amphiphilic layer. A salt gradient is disclosed in Holden et al., J Am

Chem Soc. 2007 Jul l l; 129(27):8650-5.

In some instances, the current passing through the pore as the polynucleotide moves with respect to the pore is used to determine the sequence of the target polynucleotide. This is Strand

Sequencing.

The method has several advantages. First, the inventors have surprisingly shown that RecD helicases have a surprisingly high salt tolerance and so the method of the invention may be carried out at high salt concentrations. In the context of Strand Sequencing, a charge carrier, such as a salt, is necessary to create a conductive solution for applying a voltage offset to capture and translocate the target polynucleotide and to measure the resulting sequence-dependent current changes as the polynucleotide moves with respect to the pore. Since the measurement signal is dependent on the concentration of the salt, it is advantageous to use high salt concentrations to increase the magnitude of the acquired signal. High salt concentrations provide a high signal to noise ratio and allow for currents indicative of the presence of a nucleotide to be identified against the background of normal current fluctuations. For Strand Sequencing, salt concentrations in excess of 100 mM are ideal, for example salt concentrations in excess of 400mM, 600mM or 800mM. The inventors have surprisingly shown that RecD helicases will function effectively at very high salt concentrations such as, for example, 1 M. The invention encompasses helicases which function effectively at salt concentrations in excess of 1M, for example 2M.

Second, when a voltage is applied, RecD helicases can surprisingly move the target polynucleotide in two directions, namely with or against the field resulting from the applied voltage. Hence, the method of the invention may be carried out in one of two preferred modes. Different signals are obtained depending on the direction the target polynucleotide moves with respect to the pore, ie in the direction of or against the field. This is discussed in more detail below.

Third, RecD helicases typically move the target polynucleotide through the pore one nucleotide at a time. RecD helicases can therefore function like a single-base ratchet. This is of course advantageous when sequencing a target polynucleotide because substantially all, if not all, of the nucleotides in the target polynucleotide may be identified using the pore.

Fourth, RecD helicases are capable of controlling the movement of single stranded polynucleotides and double stranded polynucleotides. This means that a variety of different target polynucleotides can be characterised in accordance with the invention.

Fifth, RecD helicases appear very resistant to the field resulting from applied voltages. The inventors have seen very little movement of the polynucleotide under an "unzipping" condition. Unzipping conditions will typically be in the absence of nucleotides, for example the absence of ATP. When the helicase is operating in unzipping mode it acts like a brake preventing the target sequence from moving through the pore too quickly under the influence of the applied voltage. This is important because it means that there are no complications from unwanted "backwards" movements when moving polynucleotides against the field resulting from an applied voltage.

Sixth, RecD helicases are easy to produce and easy to handle. Their use therefore contributed to a straightforward and less expensive method of sequencing.

The method of the invention is for characterising a target polynucleotide. A

polynucleotide, such as a nucleic acid, is a macromolecule comprising two or more nucleotides. The polynucleotide or nucleic acid may comprise any combination of any nucleotides. The nucleotides can be naturally occurring or artificial. One or more nucleotides in the target polynucleotide can be oxidized or methylated. One or more nucleotides in the target polynucleotide may be damaged. One or more nucleotides in the target polynucleotide may be modified, for instance with a label or a tag. The target polynucleotide may comprise one or more spacers.

A nucleotide typically contains a nucleobase, a sugar and at least one phosphate group. The nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines and more specifically adenine, guanine, thymine, uracil and cytosine. The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The nucleotide is typically a ribonucleotide or deoxyribonucleotide. The nucleotide typically contains a monophosphate, diphosphate or triphosphate. Phosphates may be attached on the 5' or 3' side of a nucleotide.

Nucleotides include, but are not limited to, adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), cytidine monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine

monophosphate (dUMP) and deoxycytidine monophosphate (dCMP). The nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP.

A nucleotide may be abasic (i.e. lack a nucleobase).

The polynucleotide may be single stranded or double stranded. At least a portion of the polynucleotide is preferably double stranded.

The polynucleotide can be a nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The target polynucleotide can comprise one strand of RNA hybridized to one strand of DNA. The polynucleotide may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA) or other synthetic polymers with nucleotide side chains.

The whole or only part of the target polynucleotide may be characterised using this method. The target polynucleotide can be any length. For example, the polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400 or at least 500 nucleotide pairs in length. The polynucleotide can be 1000 or more nucleotide pairs, 5000 or more nucleotide pairs in length or 100000 or more nucleotide pairs in length.

The target polynucleotide is present in any suitable sample. The invention is typically carried out on a sample that is known to contain or suspected to contain the target

polynucleotide. Alternatively, the invention may be carried out on a sample to confirm the identity of one or more target polynucleotides whose presence in the sample is known or expected. The sample may be a biological sample. The invention may be carried out in vitro on a sample obtained from or extracted from any organism or microorganism. The organism or microorganism is typically archaean, prokaryotic or eukaryotic and typically belongs to one the five kingdoms: plantae, animalia, fungi, monera and protista. The invention may be carried out in vitro on a sample obtained from or extracted from any virus. The sample is preferably a fluid sample. The sample typically comprises a body fluid of the patient. The sample may be urine, lymph, saliva, mucus or amniotic fluid but is preferably blood, plasma or serum. Typically, the sample is human in origin, but alternatively it may be from another mammal animal such as from commercially farmed animals such as horses, cattle, sheep or pigs or may alternatively be pets such as cats or dogs. Alternatively a sample of plant origin is typically obtained from a commercial crop, such as a cereal, legume, fruit or vegetable, for example wheat, barley, oats, canola, maize, soya, rice, bananas, apples, tomatoes, potatoes, grapes, tobacco, beans, lentils, sugar cane, cocoa, cotton.

The sample may be a non-biological sample. The non-biological sample is preferably a fluid sample. Examples of a non-biological sample include surgical fluids, water such as drinking water, sea water or river water, and reagents for laboratory tests.

The sample is typically processed prior to being assayed, for example by centrifugation or by passage through a membrane that filters out unwanted molecules or cells, such as red blood cells. The sample may be measured immediately upon being taken. The sample may also be typically stored prior to assay, preferably below -70°C.

A transmembrane pore is a structure that crosses the membrane to some degree. It permits ions, such as hydrated ions, driven by an applied potential to flow across or within the membrane. The transmembrane pore typically crosses the entire membrane so that ions may flow from one side of the membrane to the other side of the membrane. However, the transmembrane pore does not have to cross the membrane. It may be closed at one end. For instance, the pore may be a well in the membrane along which or into which ions may flow.

Any membrane may be used in accordance with the invention. Suitable membranes are well-known in the art. The membrane is preferably an amphiphilic layer. An amphiphilic layer is a layer formed from amphiphilic molecules, such as phospholipids, which have both at least one hydrophilic portion and at least one lipophilic or hydrophobic portion. The amphiphilic layer may be a monolayer or a bilayer. The amphiphilic layer is typically a planar lipid bilayer or a supported bilayer.

The amphiphilic layer is typically a lipid bilayer. Lipid bilayers are models of cell membranes and serve as excellent platforms for a range of experimental studies. For example, lipid bilayers can be used for in vitro investigation of membrane proteins by single-channel recording. Alternatively, lipid bilayers can be used as biosensors to detect the presence of a range of substances. The lipid bilayer may be any lipid bilayer. Suitable lipid bilayers include, but are not limited to, a planar lipid bilayer, a supported bilayer or a liposome. The lipid bilayer is preferably a planar lipid bilayer. Suitable lipid bilayers are disclosed in International

Application No. PCT/GB08/000563 (published as WO 2008/102121), International Application No. PCT/GB08/004127 (published as WO 2009/077734) and International Application No. PCT/GB2006/001057 (published as WO 2006/100484).

Methods for forming lipid bilayers are known in the art. Suitable methods are disclosed in the Example. Lipid bilayers are commonly formed by the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA., 1972; 69: 3561-3566), in which a lipid monolayer is carried on aqueous solution/air interface past either side of an aperture which is perpendicular to that interface.

The method of Montal & Mueller is popular because it is a cost-effective and relatively straightforward method of forming good quality lipid bilayers that are suitable for protein pore insertion. Other common methods of bilayer formation include tip-dipping, painting bilayers and patch-clamping of liposome bilayers.

In a preferred embodiment, the lipid bilayer is formed as described in International Application No. PCT/GB08/004127 (published as WO 2009/077734).

In another preferred embodiment, the membrane is a solid state layer. A solid-state layer is not of biological origin. In other words, a solid state layer is not derived from or isolated from a biological environment such as an organism or cell, or a synthetically manufactured version of a biologically available structure. Solid state layers can be formed from both organic and inorganic materials including, but not limited to, microelectronic materials, insulating materials such as S1₃N4, AI2O₃, and SiO, organic and inorganic polymers such as polyamide, plastics such as Teflon® or elastomers such as two-component addition-cure silicone rubber, and glasses. The solid state layer may be formed from monatomic layers, such as graphene, or layers that are only a few atoms thick. Suitable graphene layers are disclosed in International Application No.

PCT/US2008/010637 (published as WO 2009/035647).

The method is typically carried out using (i) an artificial amphiphilic layer comprising a pore, (ii) an isolated, naturally-occurring lipid bilayer comprising a pore, or (iii) a cell having a pore inserted therein. The method is typically carried out using an artificial amphiphilic layer, such as an artificial lipid bilayer. The layer may comprise other transmembrane and/or intramembrane proteins as well as other molecules in addition to the pore. Suitable apparatus and conditions are discussed below. The method of the invention is typically carried out in vitro.

The polynucleotide may be coupled to the membrane. This may be done using any known method. If the membrane is an amphiphilic layer, such as a lipid bilayer (as discussed in detail above), the polynucleotide is preferably coupled to the membrane via a polypeptide present in the membrane or a hydrophobic anchor present in the membrane. The hydrophobic anchor is preferably a lipid, fatty acid, sterol, carbon nanotube or amino acid.

The polynucleotide may be coupled directly to the membrane. The polynucleotide is preferably coupled to the membrane via a linker. Preferred linkers include, but are not limited to, polymers, such as polynucleotides, polyethylene glycols (PEGs) and polypeptides. If a polynucleotide is coupled directly to the membrane, then some data will be lost as the characterising run cannot continue to the end of the polynucleotide due to the distance between the membrane and the helicase. If a linker is used, then the polynucleotide can be processed to completion. If a linker is used, the linker may be attached to the polynucleotide at any position. The linker is preferably attached to the polynucleotide at the tail polymer.

The coupling may be stable or transient. For certain applications, the transient nature of the coupling is preferred. If a stable coupling molecule were attached directly to either the 5' or 3' end of a polynucleotide, then some data will be lost as the characterising run cannot continue to the end of the polynucleotide due to the distance between the bilayer and the helicase' s active site. If the coupling is transient, then when the coupled end randomly becomes free of the bilayer, then the polynucleotide can be processed to completion. Chemical groups that form stable or transient links with the membrane are discussed in more detail below. The

polynucleotide may be transiently coupled to an amphiphilic layer, such as a lipid bilayer using cholesterol or a fatty acyl chain. Any fatty acyl chain having a length of from 6 to 30 carbon atoms, such as hexadecanoic acid, may be used.

In preferred embodiments, the polynucleotide is coupled to an amphiphilic layer.

Coupling of polynucleotides to synthetic lipid bilayers has been carried out previously with various different tethering strategies. These are summarised in Table 1 below.

Table 1

Attachment group Type of coupling Reference

Thiol Stable Yoshina-Ishii, C. and S. G. Boxer (2003). "Arrays of mobile tethered vesicles on supported lipid bilayers." J Am Chem Soc 125(13): 3696-7.

Biotin Stable Nikolov, V., R. Lipowsky, et al. (2007). "Behavior of giant vesicles with anchored DNA molecules." Biophvs J 92(12): 4356-68

Cholestrol Transient Pfeiffer, I. and F. Hook (2004). "Bivalent cholesterol- based coupling of oligonucletides to lipid membrane assemblies." J Am Chem Soc 126(33): 10224-5

Lipid Stable van Lengerich, B , R. J. Rawle, et al. "Covalent attachment of lipid vesicles to a fluid-supported bilayer allows observation of DNA-mediated vesicle interactions." Langmuir 26(11): 8666-72

Polynucleotides may be functionalized using a modified phosphoramidite in the synthesis reaction, which is easily compatible for the addition of reactive groups, such as thiol, cholesterol, lipid and biotin groups. These different attachment chemistries give a suite of attachment options for polynucleotides. Each different modification group tethers the polynucleotide in a slightly different way and coupling is not always permanent so giving different dwell times for the polynucleotide to the bilayer. The advantages of transient coupling are discussed above.

Coupling of polynucleotides can also be achieved by a number of other means provided that a reactive group can be added to the polynucleotide. The addition of reactive groups to either end of DNA has been reported previously. A thiol group can be added to the 5' of ssDNA using polynucleotide kinase and ATPyS (Grant, G. P. and P. Z. Qin (2007). "A facile method for attaching nitroxide spin labels at the 5' terminus of nucleic acids. " Nucleic Acids Res 35(10): e77). A more diverse selection of chemical groups, such as biotin, thiols and fluorophores, can be added using terminal transferase to incorporate modified oligonucleotides to the 3 ' of ssDNA (Kumar, A., P. Tchen, et al. (1988). "Nonradioactive labeling of synthetic oligonucleotide probes with terminal deoxynucleotidyl transferase." Anal Biochem 169(2): 376-82).

Alternatively, the reactive group could be considered to be the addition of a short piece of DNA complementary to one already coupled to the bilayer, so that attachment can be achieved via hybridisation. Ligation of short pieces of ssDNA have been reported using T4 RNA ligase I (Troutt, A. B., M. G. McHeyzer- Williams, et al. (1992). "Ligation-anchored PCR: a simple amplification technique with single-sided specificity." Proc Natl Acad Sci U S A 89(20): 9823- 5). Alternatively either ssDNA or dsDNA could be ligated to native dsDNA and then the two strands separated by thermal or chemical denaturation. To native dsDNA, it is possible to add either a piece of ssDNA to one or both of the ends of the duplex, or dsDNA to one or both ends. Then, when the duplex is melted, each single strand will have either a 5' or 3 ' modification if ssDNA was used for ligation or a modification at the 5' end, the 3 ' end or both if dsDNA was used for ligation. If the polynucleotide is a synthetic strand, the coupling chemistry can be incorporated during the chemical synthesis of the polynucleotide. For instance, the

polynucleotide can be synthesized using a primer a reactive group attached to it.

A common technique for the amplification of sections of genomic DNA is using polymerase chain reaction (PCR). Here, using two synthetic oligonucleotide primers, a number of copies of the same section of DNA can be generated, where for each copy the 5' of each strand in the duplex will be a synthetic polynucleotide. By using an antisense primer that has a reactive group, such as a cholesterol, thiol, biotin or lipid, each copy of the target DNA amplified will contain a reactive group for coupling.

The transmembrane pore is preferably a transmembrane protein pore. A transmembrane protein pore is a protein structure that crosses the membrane to some degree. It permits ions driven by an applied potential to flow across or within the membrane. A transmembrane protein pore is typically a polypeptide or a collection of polypeptides that permits ions, such as analyte, to flow from one side of a membrane to the other side of the membrane. However, the transmembrane protein pore does not have to cross the membrane. It may be closed at one end. For instance, the transmembrane pore may form a well in the membrane along which or into which ions may flow. The transmembrane protein pore preferably permits analytes, such as nucleotides, to flow across or within the membrane. The transmembrane protein pore allows a polynucleotide, such as DNA or RNA, to be moved through the pore.

The transmembrane protein pore may be a monomer or an oligomer. The pore is preferably made up of several repeating subunits, such as 6, 7, 8 or 9 subunits. The pore is preferably a hexameric, heptameric, octameric or nonameric pore.

The transmembrane protein pore typically comprises a barrel or channel through which the ions may flow. The subunits of the pore typically surround a central axis and contribute strands to a transmembrane β barrel or channel or a transmembrane oc-helix bundle or channel.

The barrel or channel of the transmembrane protein pore typically comprises amino acids that facilitate interaction with analyte, such as nucleotides, polynucleotides or nucleic acids. These amino acids are preferably located near a constriction of the barrel or channel. The transmembrane protein pore typically comprises one or more positively charged amino acids, such as arginine, lysine or histidine, or aromatic amino acids, such as tyrosine or tryptophan. These amino acids typically facilitate the interaction between the pore and nucleotides, polynucleotides or nucleic acids.

Transmembrane protein pores for use in accordance with the invention can be derived from β-barrel pores or a-helix bundle pores, β-barrel pores comprise a barrel or channel that is formed from β-strands. Suitable β-barrel pores include, but are not limited to, β-toxins, such as oc-hemolysin, anthrax toxin and leukocidins, and outer membrane proteins/porins of bacteria, such as, Mycobacterium smegmatis porin (Msp), for example MspA, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A and Neisseria autotransporter lipoprotein (NalP). a-helix bundle pores comprise a barrel or channel that is formed from a-helices. Suitable a-helix bundle pores include, but are not limited to, inner membrane proteins and a outer membrane proteins, such as WZA and ClyA toxin. The transmembrane pore may be derived from Msp or from a-hemolysin (a-HL).

The transmembrane protein pore is preferably derived from Msp, preferably from MspA. Such a pore will be oligomeric and typically comprises 7, 8, 9 or 10 monomers derived from Msp. The pore may be a homo-oligomeric pore derived from Msp comprising identical monomers. Alternatively, the pore may be a hetero-oligomeric pore derived from Msp comprising at least one monomer that differs from the others. Preferably the pore is derived from MspA or a homolog or paralog thereof.

A monomer derived from Msp comprises the sequence shown in SEQ ID NO: 2 or a variant thereof. SEQ ID NO: 2 is the MS-(B 1)8 mutant of the MspA monomer. It includes the following mutations: D90N, D91N, D93N, Dl 18R, D134R and E139K. A variant of SEQ ID NO: 2 is a polypeptide that has an amino acid sequence which varies from that of SEQ ID NO: 2 and which retains its ability to form a pore. The ability of a variant to form a pore can be assayed using any method known in the art. For instance, the variant may be inserted into an amphiphilic layer along with other appropriate subunits and its ability to oligomerise to form a pore may be determined. Methods are known in the art for inserting subunits into membranes, such as amphiphilic layers. For example, subunits may be suspended in a purified form in a solution containing a lipid bilayer such that it diffuses to the lipid bilayer and is inserted by binding to the lipid bilayer and assembling into a functional state. Alternatively, subunits may be directly inserted into the membrane using the "pick and place" method described in M.A.

Holden, H. Bayley. J. Am. Chem. Soc. 2005, 127, 6502-6503 and International Application No. PCT/GB2006/001057 (published as WO 2006/100484).

Over the entire length of the amino acid sequence of SEQ ID NO: 2, a variant will preferably be at least 50% homologous to that sequence based on amino acid identity. More preferably, the variant may be at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of SEQ ID NO: 2 over the entire sequence. There may be at least 80%, for example at least 85%, 90% or 95%, amino acid identity over a stretch of 100 or more, for example 125, 150, 175 or 200 or more, contiguous amino acids ("hard homology").

Standard methods in the art may be used to determine homology. For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology, for example used on its default settings (Devereux et al (1984) Nucleic Acids Research 12, p387- 395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent residues or corresponding sequences (typically on their default settings)), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S.F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information

(http://www.ncbi.nlm.nih.gov/).

SEQ ID NO: 2 is the MS-(B 1)8 mutant of the MspA monomer. The variant may comprise any of the mutations in the MspB, C or D monomers compared with MspA. The mature forms of MspB, C and D are shown in SEQ ID NOs: 5 to 7. In particular, the variant may comprise the following substitution present in MspB: A138P. The variant may comprise one or more of the following substitutions present in MspC: A96G, N102E and A138P. The variant may comprise one or more of the following mutations present in MspD: Deletion of Gl, L2V, E5Q, L8V, D13G, W21A, D22E, K47T, I49H, I68V, D91G, A96Q, N102D, S 103T, VI 041, S136K and G141A. The variant may comprise combinations of one or more of the mutations and substitutions from Msp B, C and D. The variant preferably comprises the mutation L88N. The variant of SEQ ID NO: 2 has the mutation L88N in addition to all the mutations of MS-B1 and is called MS-B2. The pore used in the invention is preferably MS- (B2)8.

Amino acid substitutions may be made to the amino acid sequence of SEQ ID NO: 2 in addition to those discussed above, for example up to 1, 2, 3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side-chain volume. The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace. Alternatively, the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well-known in the art and may be selected in accordance with the properties of the 20 main amino acids as defined in Table 2 below. Where amino acids have similar polarity, this can also be determined by reference to the hydropathy scale for amino acid side chains in Table 3.

Table 2 - Chemical properties of amino acids

Ala aliphatic, hydrophobic, neutral Met hydrophobic, neutral

Cys polar, hydrophobic, neutral Asn polar, hydrophilic, neutral

Asp polar, hydrophilic, charged (-) Pro hydrophobic, neutral

Glu polar, hydrophilic, charged (-) Gin polar, hydrophilic, neutral

Phe aromatic, hydrophobic, neutral Arg polar, hydrophilic, charged (+) Gly aliphatic, neutral Ser polar, hydrophilic, neutral

His aromatic, polar, hydrophilic, Thr polar, hydrophilic, neutral

charged (+)

He aliphatic, hydrophobic, neutral Val aliphatic, hydrophobic, neutral

Lys polar, hydrophilic, charged(+) Trp aromatic, hydrophobic, neutral

Leu aliphatic, hydrophobic, neutral Tyr aromatic, polar, hydrophobic

Table 3- Hydropathy scale

Side Chain Hydropathy

He 4.5

Val 4.2

Leu 3.8

Phe 2.8

Cys 2.5

Met 1.9

Ala 1.8

Gly -0.4

Thr -0.7

Ser -0.8

Trp -0.9

Tyr -1.3

Pro -1.6

His -3.2

Glu -3.5

Gin -3.5

Asp -3.5

Asn -3.5

Lys -3.9

Arg -4.5

One or more amino acid residues of the amino acid sequence of SEQ ID NO: 2 may additionally be deleted from the polypeptides described above. Up to 1, 2, 3, 4, 5, 10, 20 or 30 residues may be deleted, or more.

Variants may include fragments of SEQ ID NO: 2. Such fragments retain pore forming activity. Fragments may be at least 50, 100, 150 or 200 amino acids in length. Such fragments may be used to produce the pores. A fragment preferably comprises the pore forming domain of SEQ ID NO: 2. Fragments must include one of residues 88, 90, 91, 105, 1 18 and 134 of SEQ ID NO: 2. Typically, fragments include all of residues 88, 90, 91, 105, 1 18 and 134 of SEQ ID NO: 2.

One or more amino acids may be alternatively or additionally added to the polypeptides described above. An extension may be provided at the amino terminal or carboxy terminal of the amino acid sequence of SEQ ID NO: 2 or polypeptide variant or fragment thereof. The extension may be quite short, for example from 1 to 10 amino acids in length. Alternatively, the extension may be longer, for example up to 50 or 100 amino acids. A carrier protein may be fused to an amino acid sequence according to the invention. Other fusion proteins are discussed in more detail below.

As discussed above, a variant is a polypeptide that has an amino acid sequence which varies from that of SEQ ID NO: 2 and which retains its ability to form a pore. A variant typically contains the regions of SEQ ID NO: 2 that are responsible for pore formation. The pore forming ability of Msp, which contains a β-barrel, is provided by β-sheets in each subunit A variant of SEQ ID NO: 2 typically comprises the regions in SEQ ID NO: 2 that form β-sheets. One or more modifications can be made to the regions of SEQ ID NO: 2 that form β-sheets as long as the resulting variant retains its ability to form a pore. A variant of SEQ ID NO: 2 preferably includes one or more modifications, such as substitutions, additions or deletions, within its oc-helices and/or loop regions.

The monomers derived from Msp may be modified to assist their identification or purification, for example by the addition of histidine residues (a hist tag), aspartic acid residues (an asp tag), a streptavidin tag or a flag tag, or by the addition of a signal sequence to promote their secretion from a cell where the polypeptide does not naturally contain such a sequence. An alternative to introducing a genetic tag is to chemically react a tag onto a native or engineered position on the pore. An example of this would be to react a gel-shift reagent to a cysteine engineered on the outside of the pore. This has been demonstrated as a method for separating hemolysin hetero-oligomers (Chem Biol. 1997 Jul; 4(7):497-505).

The monomer derived from Msp may be labelled with a revealing label. The revealing label may be any suitable label which allows the pore to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, e.g. ¹²⁵1, ⁵ S, enzymes, antibodies, antigens, polynucleotides and ligands such as biotin.

The monomer derived from Msp may also be produced using D-amino acids. For instance, the monomer derived from Msp may comprise a mixture of L-amino acids and D- amino acids. This is conventional in the art for producing such proteins or peptides.

The monomer derived from Msp contains one or more specific modifications to facilitate nucleotide discrimination. The monomer derived from Msp may also contain other non-specific modifications as long as they do not interfere with pore formation. A number of non-specific side chain modifications are known in the art and may be made to the side chains of the monomer derived from Msp. Such modifications include, for example, reductive alkylation of amino acids by reaction with an aldehyde followed by reduction with NaBFL, amidination with methylacetimidate or acylation with acetic anhydride.

The monomer derived from Msp can be produced using standard methods known in the art. The monomer derived from Msp may be made synthetically or by recombinant means. For example, the pore may be synthesized by in vitro translation and transcription (IVTT). Suitable methods for producing pores are discussed in International Application Nos. PCT/GB09/001690 (published as WO 2010/004273), PCT/GB09/001679 (published as WO 2010/004265) or PCT/GB 10/000133 (published as WO 2010/086603). Methods for inserting pores into membranes are discussed.

The transmembrane protein pore is also preferably derived from a-hemolysin (a-HL).

The wild type a-HL pore is formed of seven identical monomers or subunits (i.e. it is heptameric). The sequence of one monomer or subunit of a-hemolysin-NN is shown in SEQ ID NO: 4. The transmembrane protein pore preferably comprises seven monomers each comprising the sequence shown in SEQ ID NO: 4 or a variant thereof. Amino acids 1, 7 to 21, 31 to 34, 45 to 51, 63 to 66, 72, 92 to 97, 104 to 111, 124 to 136, 149 to 153, 160 to 164, 173 to 206, 210 to 213, 217, 218, 223 to 228, 236 to 242, 262 to 265, 272 to 274, 287 to 290 and 294 of SEQ ID NO: 4 form loop regions. Residues 113 and 147 of SEQ ID NO: 4 form part of a constriction of the barrel or channel of a-HL.

In such embodiments, a pore comprising seven proteins or monomers each comprising the sequence shown in SEQ ID NO: 4 or a variant thereof are preferably used in the method of the invention. The seven proteins may be the same (homoheptamer) or different

(heteroheptamer).

A variant of SEQ ID NO: 4 is a protein that has an amino acid sequence which varies from that of SEQ ID NO: 4 and which retains its pore forming ability. The ability of a variant to form a pore can be assayed using any method known in the art. For instance, the variant may be inserted into an amphiphilic layer, such as a lipid bilayer, along with other appropriate subunits and its ability to oligomerise to form a pore may be determined. Methods are known in the art for inserting subunits into amphiphilic layers, such as lipid bilayers. Suitable methods are discussed above.

The variant may include modifications that facilitate covalent attachment to or interaction with the helicase. The variant preferably comprises one or more reactive cysteine residues that facilitate attachment to the helicase. For instance, the variant may include a cysteine at one or more of positions 8, 9, 17, 18, 19, 44, 45, 50, 51, 237, 239 and 287 and/or on the amino or carboxy terminus of SEQ ID NO: 4. Preferred variants comprise a substitution of the residue at position 8, 9, 17, 237, 239 and 287 of SEQ ID NO: 4 with cysteine (A8C, T9C, N17C, K237C, S239C or E287C). The variant is preferably any one of the variants described in International Application No. PCT/GB09/001690 (published as WO 2010/004273), PCT/GB09/001679 (published as WO 2010/004265) or PCT/GB 10/000133 (published as WO 2010/086603).

The variant may also include modifications that facilitate any interaction with nucleotides.

The variant may be a naturally occurring variant which is expressed naturally by an organism, for instance by a Staphylococcus bacterium. Alternatively, the variant may be expressed in vitro or recombinantly by a bacterium such as Escherichia coli. Variants also include non-naturally occurring variants produced by recombinant technology. Over the entire length of the amino acid sequence of SEQ ID NO: 4, a variant will preferably be at least 50% homologous to that sequence based on amino acid identity. More preferably, the variant polypeptide may be at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of SEQ ID NO: 4 over the entire sequence. There may be at least 80%, for example at least 85%, 90% or 95%, amino acid identity over a stretch of 200 or more, for example 230, 250, 270 or 280 or more, contiguous amino acids ("hard homology"). Homology can be determined as discussed above.

Amino acid substitutions may be made to the amino acid sequence of SEQ ID NO: 4 in addition to those discussed above, for example up to 1, 2, 3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions may be made as discussed above.

One or more amino acid residues of the amino acid sequence of SEQ ID NO: 4 may additionally be deleted from the polypeptides described above. Up to 1, 2, 3, 4, 5, 10, 20 or 30 residues may be deleted, or more.

Variants may be fragments of SEQ ID NO: 4. Such fragments retain pore-forming activity. Fragments may be at least 50, 100, 200 or 250 amino acids in length. A fragment preferably comprises the pore-forming domain of SEQ ID NO: 4. Fragments typically include residues 119, 121, 135. 113 and 139 of SEQ ID NO: 4.

One or more amino acids may be alternatively or additionally added to the polypeptides described above. An extension may be provided at the amino terminus or carboxy terminus of the amino acid sequence of SEQ ID NO: 4 or a variant or fragment thereof. The extension may be quite short, for example from 1 to 10 amino acids in length. Alternatively, the extension may be longer, for example up to 50 or 100 amino acids. A carrier protein may be fused to a pore or variant

As discussed above, a variant of SEQ ID NO: 4 is a subunit that has an amino acid sequence which varies from that of SEQ ID NO: 4 and which retains its ability to form a pore. A variant typically contains the regions of SEQ ID NO: 4 that are responsible for pore formation. The pore forming ability of a-HL, which contains a β-barrel, is provided by β-strands in each subunit. A variant of SEQ ID NO: 4 typically comprises the regions in SEQ ID NO: 4 that form β-strands. The amino acids of SEQ ID NO: 4 that form β-strands are discussed above. One or more modifications can be made to the regions of SEQ ID NO: 4 that form β-strands as long as the resulting variant retains its ability to form a pore. Specific modifications that can be made to the β-strand regions of SEQ ID NO: 4 are discussed above.

A variant of SEQ ID NO: 4 preferably includes one or more modifications, such as substitutions, additions or deletions, within its -helices and/or loop regions. Amino acids that form a-helices and loops are discussed above.

The variant may be modified to assist its identification or purification as discussed above.

Pores derived from a-HL can be made as discussed above with reference to pores derived from Msp.

In some embodiments, the transmembrane protein pore is chemically modified. The pore can be chemically modified in any way and at any site. The transmembrane protein pore is preferably chemically modified by attachment of a molecule to one or more cysteines (cysteine linkage), attachment of a molecule to one or more lysines, attachment of a molecule to one or more non-natural amino acids, enzyme modification of an epitope or modification of a terminus.

Suitable methods for carrying out such modifications are well-known in the art. The

transmembrane protein pore may be chemically modified by the attachment of any molecule.

For instance, the pore may be chemically modified by attachment of a dye or a fluorophore.

Any number of the monomers in the pore may be chemically modified. One or more, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the monomers is preferably chemically modified as discussed above.

The reactivity of cysteine residues may be enhanced by modification of the adjacent residues. For instance, the basic groups of flanking arginine, histidine or lysine residues will change the pKa of the cysteines thiol group to that of the more reactive S^" group. The reactivity of cysteine residues may be protected by thiol protective groups such as dTNB. These may be reacted with one or more cysteine residues of the pore before a linker is attached.

The molecule (with which the pore is chemically modified) may be attached directly to the pore or attached via a linker as disclosed in International Application Nos.

PCT/GB09/001690 (published as WO 2010/004273), PCT/GB09/001679 (published as WO 2010/004265) or PCT/GB 10/000133 (published as WO 2010/086603). Any RecD helicase may be used in accordance with the invention. The structures of RecD helicases are known in the art (FEES J. 2008 Apr;275(8): 1835-51. Epiib 2008 Mar 9. ATPase activity of RecD is essential for growth of the Antarctic Pseudomonas syringae Lz4W at low temperature. Satapathy AK, Pavankumar XL. Bhattacharjya S, Sankaranarayanan R, Ray M ; EMS Microbiol Rev. 2009 May;33(3):657-87. The diversity of conjugative relaxases and its application in plasmid classification. Garcillan-Barcia MP, Francia MV, de la Cruz F; J Biol Chem. 201 1 Apr 8;286(14): 12670-82. Epub 201 1 Feb 2. Functional characterization of the multidomain F plasmid Tral re!axase-heiicase. Cheng Y, McNamara DE, Miley Ml, Nash RP, Redinbo MR).

The RecD helicase typical iy comprises the amino acid motif XI -X2-X3-G-X4-X5-X6-

X7 (hereinafter called the RecD-like motif I; SEQ ID NO: 8), wherein XI is G, S or A, X2 is any amino acid, X3 is P, A, S or G, X4 is T, A, V, S or C X5 is G or A, X6 is K. or R and X7 is T or S. XI is preferably G. X2 is preferably G, L Y or A. X2 is more preferably G. X3 is preferably F or A , X4 is preferably T, A, V or C. X4 is preferably T, V or C. X5 is preferably G. X6 is preferably K. X7 is preferably T or S. The RecD helicase preferably comprises Q-(X8)i6-i*-Xl - X2-X3-G-X4-X5-X6-X7 (hereinafter called the extended RecD-like motif I; SEQ ID NOs: 9, 10 and 11 where there are 16, 17 and 1 8 X8s respectively), wherein XI to X7 are as defined above and X8 is any amino acid. There are preferably 16 X8 residues (i.e. (Χ8) _>) n the extended RecD-like motif I (SEQ ID NO: 9), Suitable sequences for (X8)ie can be identified in SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 37, 39, 41 , 42 and 44.

The RecD helicase preferably comprises the amino acid motif G-G-P-G-Xa-G-K-Xb (hereinafter called the RecD motif Ϊ; SEQ ID NO: 12) wherein Xa is T, V or C and Xb is T or S. Xa is preferably T. Xb is preferably T. The Rec-D helicase preferably comprises the sequence G-G-P-G- C-G- - i (SEQ I^'D NO: 19; see Table 5). The RecD helicase more preferably comprises the amino acid motif Q-(X8)_{{ 6}-j 8-G-G-P-G-Xa-G-K-Xb (hereinafter called the extended RecD motif i; SEQ ID NOs: 13, H and 1.5 where there are 16, 17 and 18 X8s respectively), wherein Xa and Xb are as defined above and X8 is any amino acid. There are preferably 16 X8 residues (i.e. (X8)j₆) in the extended RecD motif I (SEQ ID NO: 13). Suitable sequences for (XS)_U can be identified in SEQ ID NOs: 18, 21 , 24, 25, 28, 30, 32, 35, 37, 39, 41, 42 and 44.

The RecD helicase typically comprises the amino acid motif XI -X2-X3-X4-X5-(X6)3-Q- X7 (hereinafter called the RecD-like motif V; SEQ ID NO: 16), wherein XI is Y, W or F, X2 is A, T, S, M, C or V, X3 is any amino acid, X4 is T, N or S, X5 is A, T, G, S, V or I, X6 is any amino acid and X7 is G or S. XI is preferably Y. X2 is preferably A, M, C or V. X2 is more preferabiy A . X3 is preferably I, M or L. X3 is more preferably [ or L. X4 is preferably T or S. X4 is more preferably T. X5 is preferably A, V or I. X5 is more preferably V or 1. X5 is most preferably V. (X6).? is preferably H-K-S, H-M-A, H-G-A or H-R-S. (X6>j is more preferably H- K-S. X7 is preferably G. The RecD helicase preferably comprises the amino acid motif Xa-Xb- Xc-Xd-Xe-H-K-S-Q-G (hereinafter called the RecD motif V: SEQ ID NO: 17), wherein Xa is Y, W or F, Xb s A, M, C or V, Xc s I, M or L, Xd i s T or S and Xe is V or I. Xa is preferably Y. Xb is preferably A. Xd is preferably T. Xe is preferably V. The RecD helicase preferably comprises (1) RecD-like motifs I and V (SEQ ID NOs: 8 and 12), (2) RecD motif I and RecD- like motif V (SEQ ID NOs: 12 and 16), (3) RecD motifs I and V (SEQ ID NOs: 12 and 17), (4) extended RecD-like motif I and RecD-like motif V (SEQ ID NOs: 9, 10 or 1 1 and 16), (5) extended RecD motif I and RecD-Like motif V (SEQ ID NOs: 13, 14 or 1 5 and 16) or (6) extended RecD motif Ϊ and RecD motif V (SEQ ID NOs: 1 , 14 or 15 and 17).

Preferred RecD motifs I are shown in Table 5 below. Preferred RecD-like motifs I are shown in Table 7 below. Preferred RecD-like motifs V are shown in Tables 5 and 7 below.

The RecD helicase is preferably one of the helicases shown in Table 4 below or a variant thereof.

Table 4 - Preferred RecD helicases and their Accession numbers

1 NP 295625.1 exodeoxvribonuclease V subunit RecD TDeinococcus radiodurans Rll

2 V P 604297.1 helicase RecD/TraA TDeinococcus geothermalis DSM 113001

3 YP 002786343.1 exodeoxvribonuclease V subunit aloha TDeinococcus deserti VCD 1151

4 3E1 S A Chain A, Structure Of An N-Terminal Truncation Of Deinococcus

5 YP 004256144.1 helicase, RecD/TraA familv TDeinococcus proteolvticus MRP1

6 YP 004170918.1 helicase, RecD/TraA familv TDeinococcus maricopensis DSM 2121 11

7 YP 004256838.1 helicase, RecD/TraA familv TDeinococcus proteolvticus MRP1

8 YP 003885838, 1 helicase, RecD/TraA familv iCyanothece sp. PCC 78221

9 ZP 08579275.1 helicase. RecD/TraA familv iPrevotella multisaccharivorax DSM 171281

10 YP 002377692, 1 helicase, RecD/TraA familv TCvanothece sp. PCC 74241

11 YP 001519318.1 RecD/TraA familv helicase TAcarvochloris marina MBIC l 10171

12 YP 003318882.1 helicase, RecD/TraA familv TSphaerobacter thermophilus DSM 207451

13 YP 004671137.1 hypothetical protein S E A07690 TSimkania negevensis Zl

14 YP 375364.1 helicase RecD/TraA rChlorobium luteolum DSM 2731 >gb I ABB24321. i l

15 YP 002418908.1 RecD/TraA familv helicase rMethvlobacterium chloromethanicum CM41

16 YP 003065757.1 Helicase rMethvlobacterium extorauens DM41 >emblCAX21689.1 |

17 ZP 00518989.1 Helicase RecD/TraA rCrocosphaera watsonii WH 85011

18 ZP 06973397.1 helicase, RecD/TraA familv [Ktedonobacter racemifer DSM 449631

19 ZP 08486910.1 helicase, RecD/TraA familv iMethvlomicrobium album BG81

20 YP 002015362.1 RecD/TraA familv helicase rProsthecochloris aestuarii DSM 2711

21 YP 001130786.1 RecD/TraA familv helicase rChlorobium phaeovibrioides DSM 2651

22 YP 002961258, 1 Helicase rMethvlobacterium extorauens AMll >gb|ACS37981.1 |

23 ZP 08772902.1 helicase, RecD/TraA familv TThiocapsa marina 581 11 >gb|EGV16093..11

24 YP 001637509.1 RecD/TraA familv helicase rMethvlobacterium extorauens PA11

25 ZP 02062824. 1 helicase. RecD/TraA familv rRickettsiella grvllil >gb|EDP46829.1 |

26 ZP 08768753. 1 helicase, RecD/TraA familv TThiocapsa marina 581 11 >gb|EGV20712..11

27 YP 001922739.1 helicase, RecD/TraA familv rMethvlobacterium populi BJ0011

28 YP 002018300.1 helicase, RecD/TraA familv TPelodictvon phaeoclathratiforme BU-11

29 ZP 06245171.1 helicase. RecD/TraA familv TVictivallis vadensis ATCC BAA-5481

30 ZP 08771217.1 helicase, RecD/TraA familv TThiocapsa marina 581 11 >gb|EGV17897.

31 ZP 08769899.1 helicase, RecD/TraA familv TThiocapsa marina 581 11 >gb|EGV18833.

32 ZP 03727363.1 Exodeoxvribonuclease V iOpitutaceae bacterium TAV21

33 ZP 05027797, 1 helicase. RecD/TraA familv TMicrocoleus chthonoplastes PCC 74201

34 YP 001521445.1 RecD/TraA familv helicase rAcarvochloris marina MBIC l 10171

35 YP 002606149.1 RecD3 rDesulfobacterium autotrophicum HRM21 >gb|ACN17985.1 |

36 YP 003165615.1 helicase. RecD/TraA familv TCandidatus Accumulibacter phosphatis

37 ZP 01732265. 1 Helicase RecD/TraA iCvanothece sp. CCY01 101 >gb|EAZ88318.1 |

YP 901533.1 RecD/TraA family helicase TPelobacter propionicus DSM 23791 YP 004121205.1 helicase, RecD/TraA family [Desulfovibrio aespoeensis Aspo-21 YP 91 1313.1 RecD/TraA family helicase TChlorobium phaeobacteroides DSM 2661 YP 002424008.1 RecD/TraA family helicase rMethylobacterium chloromethanicum YP 320143.1 helicase RecD/TraA [Anabaena variabilis ATCC 294131

Y P 001603050.1 exodeoxvribonuclease TGluconacetobacter diazotrophicus PA1 51 ZP 05054956, 1 helicase, RecD/TraA family lOctadecabacter antarcticus 3071

YP 003445164.1 helicase, RecD/TraA family rAllochromatium vinosum DSM 1801 NP 4901 77.1 exodeoxvribonuclease V, alpha chain TNostoc sp. PCC 71201

NP 923575.1 exodeoxvribonuclease V alpha chain TGloeobacter violaceus PCC 74211 YP 001601244.1 exodeoxyribonuclease V alpha chain [Gluconacetobacter diazotrophicus YP 004748470.1 exodeoxvribonuclease V subunit alpha rAcidithiobacillus caldus SM-11 Y P 004863326.1 helicase , RecD/TraA family TCandidatus Chloracidobacterium

YP 001520750.1 RecD/TraA family helicase rAcaryochloris marina MBICl 10171 YP 003197384.1 helicase, RecD/TraA family [Desulfohalobium retbaense DSM 56921 ZP 08900128.1 helicase, RecD/TraA family protein rGluconacetobacter oboediens YP 002275391.1 helicase, RecD/TraA family TGluconacetobacter diazotrophicus PA1 51 YP 003156740.1 RecD/TraA family helicase TDesulfomicrobium baculatum DSM 40281 ZP 08821817.1 helicase, RecD/TraA family I hiorhodococcus drewsii AZ 11

ZP 01731986, 1 Helicase RecD/TraA TCyanothece sp. CCY01 101 >gb|EAZ88625.1 YP 001943002.1 RecD/TraA family helicase TChlorobium limicola DSM 2451

ZP 083 18929.1 hypothetical protein SXCC 04894 rGluconacetobacter sp. SXCC-H YP 002017890.1 helicase, RecD/TraA family [Pelodictyon phaeoclathratiforme BU-11 ZP 07972826.1 RecD/TraA family helicase TSynechococcus sp. CB01011

YP 003189342.1 DNA helicase RecD/TraA [Acetobacter pasteurianus ΠΌ 3283-011 YP 001959197.1 RecD/TraA family helicase TChlorobium phaeobacteroides BS 11 ZP 05064957, 1 helicase, RecD/TraA family iOctadecabacter antarcticus 2381

YP 001772290.1 RecD/TraA family helicase rMethylobacterium sp. 4-461

YP 001998378.1 RecD/TraA family helicase [Chlorobaculum parvum NCIB 83271 YP 001869949.1 RecD/TraA family helicase TNostoc punctiforme PCC 731021

ZP 08109907.1 helicase, RecD/TraA family rDesulfovibrio sp. ND1321

ZP 06965850. 1 helicase, RecD/TraA family [Ktedonobacter racemifer DSM 449631 ZP 05428586.1 helicase, RecD/TraA family [Clostridium thermocellum DSM 23601 ZP 05404007, 1 helicase, RecD/TraA family TMitsuokella multacida DSM 205441 YP 002992028.1 helicase, RecD/TraA family rDesulfovibrio salexigens DSM 26381 ZP 02190744.1 Helicase RecD/TraA Talpha proteobacterium BAL1991

ZP 08959149.1 RecD/TraA family helicase THalomonas sp. HAL 11 >gb|EHA16215.1 | YP 003709145.1 exodeoxyribonuclease V, alpha subunit TWaddlia chondrophila WSU YP 003528424.1 helicase, RecD/TraA family fNitrosococcus halophilus Nc41

ZP 02191403.1 Helicase RecD/TraA [alpha proteobacterium BAL1991

Y P 004802608.1 helicase, RecD/TraA family [Streptomyces sp. SirexAA-El

CCB91 170.1 uncharacterized protein yrrC [Waddlia chondrophila 2032/991

YP 28981 1.1 helicase RecD/TraA TThermobifida fusca YX1 >gb|AAZ55788.1 | ZP 07015918.1 helicase, RecD/TraA family [Desulfonatronospira thiodismutans AS03- YP 004766648.1 helicase TMegasphaera elsdenii DSM 204601 >emb|CCC73821.11 ZP 04708454.1 putative exodeoxyribonuclease V [Streptomyces roseosporus NRRL YP 001039578.1 RecD/TraA family helicase [Clostridium thermocellum ATCC 274051 YP 594664.1 exonuclease V subunit alpha fLawsonia intracellularis PHE/MNl-001 NP 662288.1 exodeoxvribonuclease V, alpha subunit, putative TChlorobium tepidum ZP 08423994.1 helicase, RecD/TraA family rDesulfovibrio africanus str. Walvis Bayl YP 007688.1 putative exodeoxyribonuclease V iCandidatus Protochlamydia

YP 002953244.1 helicase RecD/TraA family protein rDesulfovibrio magneticus RS-11 ADW05584.1 helicase, RecD/TraA family [Streptomyces flavogriseus ATCC 333311 ZP 01385982, 1 Helicase RecD/TraA TChlorobium ferrooxidans DSM 130311

YP 001716965.1 RecD/TraA family helicase TCandidatus Desulforudis audaxviator ADL25833.1 helicase, RecD/TraA family iFibrobacter succinogenes subsp.

YP 002480970.1 helicase, RecD/TraA family TCyanothece sp. PCC 74251

YP 004516136.1 helicase, RecD/TraA family TDesulfotomaculum kuznetsovii DSM ZP 08778308.1 exodeoxyribonuclease iCandidatus Odyssella thessalonicensis L131 ZP 06825719 RecD/TraA family helicase rStreptomyces so. SPB741 >gb|EDY42267.2| ZP 05293745 Exodeoxvribonuclease V alpha chain TAcidithiobacillus caldus ATCC

YP 480657. 1 helicase RecD/TraA TFrankia sp. CcI31 >gb|ABD10928.1 | Helicase

ZP 07017628 helicase, RecD/TraA familv TDesulfonatronospira thiodismutans AS03-11 YP 379155.1 helicase RecD/TraA TChlorobium chlorochromatii CaD31

YP 004897355.1 helicase TAcidaminococcus intestini RyC-MR951 >gb I AEQ23215. i l ZP 03311944. 1 hypothetical protein DESPIG 01864 [Desulfovibrio piger ATCC 290981

YP 004783252.1 RecD/TraA family helicase TAcidithiobacillus ferrivorans SS31

03928493. 1 helicase TAcidaminococcus sp. D211 >gb|EEH89723.1 | helicase

06530901. 1 RecD/TraA family helicase TStreptomyces lividans TK241

01667371. 1 helicase, RecD/TraA family I hermosinus carboxydivorans Nor 11 0894:2446. 1 helicase, RecD/TraA family I hiorhodovibrio sp. 9701 >gb|EGZ54636.1 |

MP 626969.1 deoxyribonuclease TStreptomyces coelicolor A3 f 2)1 >emb|CAB66276.1 ADU73817.1 helicase, RecD/TraA family [Clostridium thermocellum DSM 13131 YP 001157093.1 RecD/TraA family helicase TSalinispora tropica CNB-4401

ZP 02929767.1 putative exodeoxvribonuclease rVerrucomicrobium spinosum DSM 41361 ZP 08455023.1 putative exodeoxvribonuclease V TStreptomyces sp. Tu60711

YP 003022840.1 helicase, RecD/TraA family [Geobacter sp. M211 >gb|ACT19082.1 |

YP 003549103 , 1 helicase, RecD/TraA familv iCoraliomargarita akaiimensis DSM 452211 YP 001530229, 1 RecD/TraA family helicase TDesulfococcus oleovorans Hxd31

YP 004461 132.1 helicase, RecD/TraA family I epidanaerobacter sp. Rell

ZP 08943153.1 helicase, RecD/TraA family IThiorhodovibrio sp. 9701 >gb|EGZ54097.1 | ZP 06560617.1 helicase, RecD/TraA familv TMegasphaera genomosp. type 1 str. 28L1 YP 002138036.1 helicase, RecD/TraA family TGeobacter bemidjiensis Beml

YP 001300657.1 exonuclease V subunit alpha TBacteroides vulgatus ATCC 84821

ZP 07303897.1 exodeoxvribonuclease V, alpha subunit TStreptomyces viridochromogenes YP 003399141.1 helicase, RecD/TraA family TAcidaminococcus fermentans DSM 207311 YP 389216.1 RecD/TraA family helicase TDesulfovibrio alaskensis G201

ZP 01085074.1 Helicase RecD/TraA TSynechococcus sp. WH 57011 >gb|EAQ75130.1 | 07271541.1 exodeoxvribonuclease V, alpha subunit TStreptomyces sp. SPB781 02731419. 1 helicase, RecD/TraA familv protein TGemmata obscuriglobus UOM 22461 08287369. 1 deoxyribonuclease TStreptomyces griseoaurantiacus M0451

CBX27215.1 hypothetical protein N47 A12440 Tuncultured Desulfobacterium sp.l YP 001530553.1 RecD/TraA family helicase TDesulfococcus oleovorans Hxd31

ZP 06707995. 1 RecD/TraA familv helicase TStreptomyces sp. el41 >sb|EFF91117.11 ZP 06917215. 1 exodeoxvribonuclease V, alpha subunit TStreptomyces sviceus ATCC YP 002955020.1 helicase RecD/TraA family protein TDesulfovibrio magneticus RS-11 ZP 07985895. 1 putative exodeoxvribonuclease V TStreptomvces sp. SA3 actFl

YP 003103858.1 helicase, RecD/TraA family iActinosynnema mirum DSM 438271 YP 001826337.1 putative exodeoxvribonuclease V TStreptomvces griseus subsp. griseus

NP 826506.1 exodeoxvribonuclease V TStreptomyces avermitilis MA-46801

ZP 01048465.1 Helicase RecD/TraA TNitrobacter sp. Nb-311A1 >gb IEA033584. i l YP 003761 94, 1 helicase, RecD/TraA familv TNitrosococcus watsonii C-l 131

YP 003681742, 1 RecD/TraA family helicase HNocardiopsis dassonvillei subsp. dassonvillei 08944617.1 helicase, RecD/TraA family IThiorhodovibrio sp. 9701 >gb|EGZ52593.1 | 08803068.1 DNA-binding protein TStreptomyces zinciresistens K421

07740397.1 helicase, RecD/TraA family TAminomonas paucivorans DSM 122601

YP 003250238.1 helicase, RecD/TraA family ITibrobacter succinogenes subsp.

01903329.1 Helicase RecD/TraA TRoseobacter sp. AzwK-3bl >gb|EDM71427.1 |

ZP 03641678.1 hypothetical protein BACCOPRO 00005 TBacteroides coprophilus DSM 06578909.1 exodeoxyribonuclease V TStreptomyces ghanaensis ATCC 146721

YP 004652609.1 protein vrrC TParachlamydia acanthamoebae UV71 >emb|CCB86755.1

ZP 06299454.1 hypothetical protein pah c032o017 TParachlamydia acanthamoebae str. YP 001771030.1 RecD/TraA family helicase rMethylobacterium sp. 4-461

ZP 08291769 1 exodeoxvribonuclease V alpha chain TChlamydophila psittaci Cal l 01 CCB74558.1 Exodeoxvribonuclease V TStreptomvces cattleva NRRL 80571

ZP 08077054 1 helicase, RecD/TraA family TPhascolarctobacterium sp. YIT 120671 ZP 07297625 RecD/TraA family helicase TStreptomyces hygroscopicus ATCC 536531 ZP 08030087 helicase, RecD/TraA familv TSelenomonas artemidis F03991 YP 003300321.1 helicase, RecD/TraA family I hermomonospora curvata DSM 431831 YP 001535192.1 RecD/TraA family helicase TSalinispora arenicola CNS-2051

NP 829514.1 RecD/TraA family helicase TChlamydophila caviae GPIC1

ZP 08843685.1 RecD/TraA family helicase TDesulfo vibrio sp. 6 1 46AFAA1

ZP 07331538.1 helicase, RecD/TraA family TDesulfovibrio fructosovorans JJ1

YP 003491438.1 DNA-binding protein TStreptomyces scabiei 87.221 >emb|CBG72898.1 | ZP 08073657. 1 helicase, RecD/TraA family TMethylocvstis sp. ATCC 492421

ZP 07829281. 1 helicase, RecD/TraA family TSelenomonas sp. oral taxon 137 str. F04301 YP 899880.1 RecD/TraA family helicase TPelobacter propionicus DSM 23791 YP 343034.1 helicase RecD/TraA TNitrosococcus oceani ATCC 197071

YP 004817633. 3 helicase, RecD/TraA family TStreptomyces violaceusniger Tu 41 131 BAJ31218.1 putative helicase RecD/TraA family protein TKitasatospora setae KM- YP 578071 .1 helicase RecD/TraA [Nitrobacter hamburgensis X141 >gb I ABE63611. i l ZP 01873510, 1 ATP-dependent exoDNAse ( exonuclease V), alpha subunit-helicase EFE27709.1 helicase, RecD/TraA family TFilifactor alocis ATCC 358961

YP 220018. 1 putative exodeoxyribonuclease rChlamydophila abortus S26/31

ZP 07327131.1 helicase, RecD/TraA family TAcetivibrio cellulolyticus CD21

YP 002480862.1 helicase. RecD/TraA family TDesulfovibrio desulfuricans subsp.

EGK69360.1 putative exodeoxyribonuclease V subunit alpha TChlamydophila abortus YP 001967390.1 helicase RecD/TraA [Rickettsia monacensisl >gb|ABQ85878.1 | helicase ZP 03754577. 1 hypothetical protein ROSEINA2194 03004 [Roseburia inulinivorans ZP 04608382. 1 helicase [Micromonospora sp. ATCC 391491 >gb|EEP74312.1 | helicase YP 001509194.1 RecD/TraA family helicase TFrankia sp. EANlpecl >gb|ABW14288.1 | ZP 06771382. 1 Exodeoxyribonuclease V TStreptomyces clavuligerus ATCC 270641 ZP 08838047.1 RecD/TraA family helicase TBilophila sp. 4 1 301 >gb|EGW42429.1 | CBE67477.1 Helicase, RecD/TraA family TNC 10 bacterium 'Dutch sediment'l YP 001950493 , 1 helicase. RecD/TraA family TGeobacter lovlevi SZ1 >gb|ACD93973.1 | ZP 02192076, 1 Helicase RecD/TraA Talpha proteobacterium BAL1991 >gb|EDP61161.11 ZP 07943852, 1 RecD/TraA family helicase TBilophila wadsworthia 3 1 61

YP 003652363.1 helicase, RecD/TraA family I hermobispora bispora DSM 438331 ZP 05005893.1 exodeoxyribonuclease V TStreptomyces clavuligerus ATCC 270641 ZP 05065242.1 helicase, RecD/TraA family TOctadecabacter antarcticus 2381

CBL24549.1 helicase, putative, RecD/TraA family TRuminococcus obeum A2-1621 YP 002499923.1 RecD/TraA family helicase rMethvlobacterium nodulans ORS 20601 YP 002432033.1 helicase, RecD/TraA family TDesulfatibacillum alkenivorans AK-011 ZP 07286835. 1 exodeoxyribonuclease V, alpha subunit TStreptomyces sp. CI

YP 001105553.1 helicase RecD/TraA TSaccharopolyspora erythraea NRRL 23381 YP 003639317.1 helicase, RecD/TraA family TThermincola sp. JR1 >gb|ADG81416.11 CBK63520.1 helicase, putative, RecD/TraA family TAlistipes shahii WAL 83011 ZP 07940306.1 RecD/TraA family helicase TBacteroides sp. 4 1 361 >gb IEFV24451. i l ZP 08905541.1 helicase RecD/TraA family protein TDesulfovibrio sp. FW1012B1 CBL07724.1 helicase, putative, RecD/TraA family TRoseburia intestinalis M50/11 ZP 03729282, 1 helicase, RecD/TraA family [Dethiobacter alkaliphilus AHT 11

YP 001220283 , 1 RecD/TraA family helicase TAcidiphilium cryptum JF-51

ZP 05382244.1 exodeoxyribonuclease V alpha chain [Chlamydia trachomatis D(s)29231 YP 001654372.1 exodeoxyribonuclease V alpha chain (^"Chlamydia trachomatis 434/Bul ZP 04743359.1 helicase, RecD/TraA family TRoseburia intestinalis Ll-821

ZP 08626355.1 helicase, RecD/TraA family protein TAcetonema longum DSM 65401 YP 004197836.1 helicase, RecD/TraA family TGeobacter sp. M181 >gb|ADW12560.1 | ZP 06415577.1 helicase, RecD/TraA family TFrankia sp. EUNlfl >gb|EFC81619.1 YP 001618568.1 exodeoxyribonuclease V TSorangium cellulosum 'So ce 56'1

ZP 05000079. 1 exodeoxyribonuclease V TStreptomyces sp. Mgll >gb|EDX24590.1 | ZP 05346027.3 helicase, RecD/TraA family TBryantella formatexigens DSM 144691 ADI10122.1 exodeoxyribonuclease V TStreptomyces bingchenggensis BCW-11 YP 001220030.1 RecD/TraA family helicase TAcidiphilium cryptum JF-51

YP 515278.1 ATP-dependent dsDNA/ssDNA exodeoxyribonuclease V alpha

ZP 04658601 , 1 exodeoxyribonuclease V alpha subunit TSelenomonas flueggei ATCC YP 002887661.1 exodeoxyribonuclease V alpha chain TChlamydia trachomatis

ADH 17723.1 exodeoxyribonuclease V alpha chain [Chlamydia trachomatis G/97681 YP 327831. 1 exodeoxvribonuclease V alpha chain [Chlamydia trachomatis A/HAR- ZP 06604245.1 RecD/TraA family helicase TSelenomonas noxia ATCC 435411

YP 004819301.1 helicase, RecD/TraA family I hermoanaerobacter wiegelii Rt8.Bll ZP 05353404.1 exodeoxvribonuclease V alpha chain [Chlamydia trachomatis 62761 YP 003340096.1 exodeoxvribonuclease V TStreptosporangium roseum DSM 430211 YP 003965592.1 Helicase RecD/TraA rPaenibacillus polymyxa SC21 >gb|AD059524.1 | CCA55822.1 RecD DNA helicase YrrC rStreptomyces venezuelae ATCC 107121 NP 296681 .1 exodeoxvribonuclease V, alpha subunit [Chlamydia muridarum Niggl YP 001126584, 1 exodeoxvribonuclease V subunit alpha [Geobacillus thermodenitrificans YP 004584034, 1 RecD/TraA family helicase [Frankia symbiont of Datisca glomeratal ZP 08710208, 1 helicase, RecD/TraA family [Megasphaera sp. UPII 135-E1

NP 219535. 1 exodeoxvribonuclease V alpha chain [Chlamydia trachomatis D/UW- ZP 05899746.1 helicase, RecD/TraA familv [Selenomonas sputigena ATCC 351851 ZP 08502002.1 RecD/TraA family helicase [Centipeda periodontii DSM 27781

ZP 06590805.1 exodeoxvribonuclease V [Streptomyces albus J10741 >gb|EFE81266.11 YP 003 6724.1 helicase, RecD/TraA family [Catenulispora acidiphila DSM 449281 YP 002953905.1 helicase RecD/TraA familv protein [Desulfovibrio magneticus RS-11 ZP 06250970. 1 helicase, RecD/TraA family [Prevotella copri DSM 182051

ZP 08634959.1 RecD/TraA family helicase [Acidiphilium sp. PM1 >gb|EG093242.1 YP 846029.1 RecD/TraA family helicase [Syntrophobacter fumaroxidans MPOBl YP 003476334, 1 helicase, RecD/TraA family [Thermoanaerobacter italicus Ab91 YP 001665762, 1 RecD/TraA familv helicase [Thermoanaerobacter pseudethanolicus YP 001662092.1 RecD/TraA family helicase [Thermoanaerobacter sp. X5141

ZP 01462693.1 helicase, RecD/TraA family [Stigmatella aurantiaca DW4/3-11

ZP 07396437.1 RecD/TraA family helicase [Selenomonas sp. oral taxon 149 str.

ZP 08211163.1 helicase, RecD/TraA family [Thermoanaerobacter ethanolicus JW 2001 YP 003953494.1 exodeoxvribonuclease v alpha chain [Stigmatella aurantiaca DW4/3-11 YP 003676322.1 RecD/TraA family helicase [Thermoanaerobacter mathranii subsp. YP 003252104.1 helicase, RecD/TraA familv [Geobacillus sp. Y412MC611

YP 001918465.1 helicase, RecD/TraA family [Natranaerobius thermophilus JW/NM-WN- ZP 08880276, 1 helicase RecD/TraA [Saccharopolyspora spinosa NRRL 183951 ZP 03991439, 1 possible exodeoxvribonuclease V alpha subunit [Oribacterium sinus Λ AG 23283. 1 probable exodeoxvribonuclease V [Saccharopolyspora spinosal

YP 148414. 1 ATP-dependent exonuclease V [Geobacillus kaustophilus HTA4261 YP 714380.1 putative exodeoxvribonuclease V [Frankia alni ACN14al

CA.⁾ 74974 I similar to exodeoxvribonuclease V alpha subunit [Candidatus Kuenenia YP 001936773.1 exodeoxvribonuclease V alpha subunit [Orientia tsutsugamushi str. YP 001248319.1 helicase RecD/TraA, ATP-dependent exoDNAse (exonuclease V) YP 004377643.1 RecD/TraA family helicase [Chlamydophila pecorum E581

CBL20603.1 helicase, putative, RecD/TraA family [Ruminococcus sp. SRI/51 YP 004421448, 1 helicase RecD/TraA [Candidatus Rickettsia amblyommii AaR/SCl ZP 04857179.1 conserved hypothetical protein [Ruminococcus sp. 5 1 39B FAA1 YP 003670549, 1 helicase, RecD/TraA family [Geobacillus sp. C56-T31 >gb|ADI25972.1 | YP 003824777.1 helicase, RecD/TraA family [Thermosediminibacter oceani DSM 166461 ZP 08812773.1 hypothetical protein DOT 4190 [Desulfosporosinus sp. OT1

ZP 07757395.1 helicase, RecD/TraA family [Megasphaera micronuciformis F03591 ZP 08131496.1 helicase, RecD/TraA family [Clostridium sp. D51 >gb|EGB91345.1 | ZP 04698234. 1 helicase, RecD/TraA family [Rickettsia endosymbiont of Ixodes AEH95290.1 putative helicase [Aplvsina aerophoba bacterial symbiont clone

V P 002464026.1 helicase, RecD/TraA family [Chloroflexus aggregans DSM 94851 YP 461625.1 exodeoxvribonuclease V subunit alpha [Syntrophus aciditrophicus SB1 YP 0101 16.1 RecD/TraA family helicase [Desulfovibrio vulgaris str. Hildenboroughl YP 001634449, 1 RecD/TraA family helicase [Chloroflexus aurantiacus J-10-fll

YP 004371330, 1 helicase, RecD/TraA family [Desulfobacca acetoxidans DSM 111091 ZP 02432140.1 hypothetical protein CLOSCI 02385 [Clostridium scindens ATCC YP 001546377.1 RecD/TraA family helicase [Herpetosiphon aurantiacus DSM 7851 ZP 01995753.1 hypothetical protein DORLON 01748 [Dorea longicatena DSM 138141 ZP 08602931 .1 RecD/TraA familv helicase [Lachnospiraceae bacterium 5 1 57FAA1 YP 003409821.1 helicase, RecD/TraA family [Geodermatophilus obscurus DSM 431601 YP 004839529.1 helicase, RecD/TraA familv protein TRoseburia hominis A2-1831

ZP 08864377.1 helicase, RecD/TraA familv TDesulfovibrio sp. A21 >gb|EGY27050.1 |

ZP 05733053.1 helicase, RecD/TraA familv TDialister invisus DSM 154701

YP 003270118.1 helicase, RecD/TraA familv iHaliangium ochraceum DSM 143651

YP 001717534.1 RecD/TraA family helicase TCandidatus Desulforudis audaxviator

YP 003317021 .1 helicase, RecD/TraA family iThermanaerovibrio acidaminovorans DSM

NP 622165.1 exonuclease V subunit alpha TThermoanaerobacter tengcongensis MB41

ZP 05346627. 1 helicase, RecD/TraA family iBryantella formatexigens DSM 144691

ZP 04451325. 1 hypothetical protein GCWU000182 00609 TAbiotrophia defectiva ATCC

NP 623674.1 exonuclease V subunit alpha I hermoanaerobacter tengcongensis MB41

ZP 02037832.1 hypothetical protein BACCAP 03451 TBacteroides capillosus ATCC

EGS35366.1 helicase, RecD/TraA familv [Finegoldia magna SY403409CC0010504171

ZP 05092205.1 helicase, RecD/TraA family rCarboxydibrachium pacificum DSM 126531

ZP 08419913.1 helicase, RecD/TraA familv TRuminococcaceae bacterium D 161

YP 001692807.1 ATP-dependent exodeoxyribonuclease subunit alpha [Finegoldia magna

ZP 07268899.1 helicase, RecD/TraA family iFinegoldia magna ACS-171-V-Col31

ZP 06598130.1 helicase, RecD/TraA family lOribacterium sp. oral taxon 078 str. F02621

YP 003807812.1 helicase, RecD/TraA familv iDesulfarculus baarsii DSM 20751

ZP 02233368.1 hypothetical protein DORFOR 00200 iDorea formicigenerans ATCC

ZP 02037912. 1 hypothetical protein BACCAP 03531 iBacteroides capillosus ATCC

ZP 07202886. 1 helicase, RecD/TraA familv idelta proteobacterium NaphS21

YP 003152507.1 helicase, RecD/TraA family iAnaerococcus prevotii DSM 205481

ZP 04861948. 1 helicase, RecD/TraA family [Clostridium botulinum D str. 18731

ZP 07398794. 1 RecD/TraA familv helicase TPeptoniphilus duerdenii ATCC BAA- 16401

YP 867272, 1 RecD/TraA familv helicase TMagnetococcus sp. MC-11

YP 003852761.1 helicase, RecD/TraA family rThermoanaerobacterium

ZP 07959337.1 RecD/TraA familv Helicase TLachnospiraceae bacterium 8 1 57FAA1

YP 004020312, 1 helicase, RecD/TraA family TFrankia sp. Eullcl >gb|ADP84442.1 |

ZP 05055731.1 helicase, RecD/TraA family rVerrucomicrobiae bacterium DG12351

YP 002936648.1 helicase, RecD/TraA family TEubacterium rectale ATCC 336561

ZP 02620578.1 helicase, RecD/TraA family [Clostridium botulinum C str. Eklundl

CB] K80090.1 helicase, putative, RecD/TraA family TCoprococcus catus GD/71

ZP 08865335.1 hypothetical protein DA2 1615 TDesulfovibrio sp. A21 >gb|EGY26242.1 |

YP 004309919.1 helicase, RecD/TraA familv [Clostridium lentocellum DSM 54271

YP 004471588.1 helicase, RecD/TraA family rThermoanaerobacterium xylanolyticum LX-

CBL21608.1 helicase, putative, RecD/TraA family TRuminococcus sp. SRI/51

YP 003844493.1 helicase, RecD/TraA family [Clostridium cellulovorans 743B1

ZP 07321245. 1 helicase, RecD/TraA family [Finegoldia magna BVS033A41

YP 699450.1 RecD/TraA family helicase [Clostridium perfringens SM1011

ZP 03781777.1 hypothetical protein RUMHYD 01213 [Blautia hydrogenotrophica DSM

YP 002435820.1 helicase, RecD/TraA familv [Desulfovibrio vulgaris str. 'Miyazaki F'l

ZP 02042737.1 hypothetical protein RUMGNA 03541 [Ruminococcus gnavus ATCC

YP 004003526, 1 helicase, recd/traa family [Caldicellulosiruptor owensensis OL1

ZP 04666257.1 helicase [Clostridials bacterium 1 7 47 FAA1 >gb|EEQ62058.1 |

YP 004025165.1 helicase, recd/traa family [Caldicellulosiruptor kronotskyensis 20021

YP 003937377.1 DNA-binding protein [Clostridium sticklandii DSM 5191

ZP 03777575.1 hypothetical protein CLOHYLEM 04627 [Clostridium hylemonae DSM

ZP 02089268.1 hypothetical protein CLOBOL 06837 [Clostridium bolteae ATCC BAA-

ZP 06946532. 1 RecD/TraA family helicase [Finegoldia magna ATCC 535161

ZP 03762681 . 1 hypothetical protein CLOSTASPAR 06723 [Clostridium asparagiforme

ZP 08933657. 1 RecD/TraA family helicase [Peptoniphilus indolicus ATCC 294271

YP 003759289.1 UvrD/REP helicase [Dehalogenimonas lykanthroporepellens BL-DC-91

ZP 02865223. 1 helicase, RecD/TraA familv [Clostridium perfringens C str. JGS 14951

YP 002315104.1 ATP-dependent exoDNAse (exonuclease V) subunit alpha - helicase

YP 003820655.1 helicase, RecD/TraA familv [Clostridium saccharolvticum WM11

NP 563091 .1 helicase, RecD/TraA family [Clostridium perfringens str. 131

ZP 02631593.1 helicase, RecD/TraA family [Clostridium perfringens E str. JGS 19871

YP 754748. 1 exodeoxyribonuclease V [Svntrophomonas wolfei subsp. wolfei str.

YP 002574552.1 RecD/TraA family helicase [Caldicellulosiruptor bescii DSM 67251 ZP 02641429.1 helicase, RecD/TraA familv [Clostridium perfringens NCTC 82391

YP 004121351.1 ATP-dependent RecD/TraA familv DNA helicase iDesulfovibrio

EGC82456.1 helicase, RecD/TraA familv TAnaerococcus prevotii ACS-065-V-Coll31

YP 004199699.1 ATP-dependent RecD/TraA familv DNA helicase TGeobacter sp. Ml 81

ZP 08616225. 1 RecD/TraA familv helicase [Lachnospiraceae bacterium 1 4 56FAA1

ZP 06113685. 1 helicase, RecD/TraA family [Clostridium hathewayi DSM 134791

ZP 0379991 1. 1 hvpothetical protein COPCOM 02174 TCoprococcus comes ATCC

YP 003841513.1 helicase, RecD/TraA family rCaldicellulosiruptor obsidiansis OB471

YP 004464342.1 RecD/TraA family ATP-dependent DNA helicase TMahella australiensis

YP 696854.1 RecD/TraA family helicase [Clostridium perfrin ens ATCC 131241

ZP 03167580.1 hvpothetical protein RUMLAC 01253 TRuminococcus lactaris ATCC

YP 847893 , 1 hypothetical protein Sfum 3789 TSyntrophobacter fumaroxidans MPOBl

ZP 05430222.1 helicase, RecD/TraA family [Clostridium thermocellum DSM 23601

ZP 02211142.1 hvpothetical protein CLOBAR 00740 [Clostridium bartlettii DSM

YP 388414.2 UvrD REP helicase iDesulfovibrio alaskensis G201 >gb|ABB38719.2|

YP 003807790.1 ATP-dependent RecD/TraA family DNA helicase [Desulfarculus baarsii

YP 003993640.1 helicase, recd/traa family [Caldicellulosiruptor hydrothermalis 1081

YP 001038644.1 ATP-dependent RecD/TraA familv DNA helicase [Clostridium

ZP 06597516.1 helicase, RecD/TraA family [Oribacterium sp. oral taxon 078 str. F02621

YP 004799933.1 helicase, RecD/TraA family [Caldicellulosiruptor lactoaceticus 6A1

YP 001179036.1 RecD/TraA family helicase [Caldicellulosiruptor saccharolyticus DSM

ZP 03759537. 1 hypothetical protein CLOSTASPAR 03561 [Clostridium asparagiforme

ZP 04564978. 1 exodeoxyribonuclease subunit V alpha [Mollicutes bacterium D71

YP 001557372.1 RecD/TraA family helicase [Clostridium phytofermentans ISDgl

ZP 02094462.1 hypothetical protein PEPMIC 01228 [Parvimonas micra ATCC 332701

ZP 02428906.1 hypothetical protein CLORAM 02328 [Clostridium ramosum DSM

ZP 07367500.1 exodeoxyribonuclease V alpha subunit [Pediococcus acidilactici DSM

YP 004027603 , 1 helicase, recd/traa family [Caldicellulosiruptor kristjanssonii 177R1B1

ZP 02420394.1 hypothetical protein ANACAC 03011 [Anaerostipes caccae DSM 146621

ZP 08707741.1 helicase, RecD/TraA family [Veillonella sp. oral taxon 780 str. F04221

ZP 08532372.1 helicase, RecD/TraA family [Caldalkalibacillus thermarum TA2.A11

YP 003119362.1 helicase, RecD/TraA family [Catenulispora acidiphila DSM 449281

YP 001821497.1 RecD/TraA family helicase rOpitutus terrae PB90-H >gb|ACB77897.1 |

YP 003427313.1 ATP-dependent exoDNAse V [Bacillus pseudofirmus OF41

ZP 07036639. 1 helicase, RecD/TraA family [Peptoniphilus sp. oral taxon 386 str. F01311

YP 004091069.1 helicase, RecD/TraA family [Ethanoligenens harbinense YUAN-31

ZP 06197663. 1 RecD/TraA family helicase [Pediococcus acidilactici 7 41

ZP 06409626. 1 helicase, RecD/TraA family [Clostridium hathewayi DSM 134791

ZP 08339411. 1 RecD/TraA family helicase [Lachnospiraceae bacterium 2 1 46FAA1

YP 004883288.1 putative nuclease [Oscillibacter valericigenes Siml8-201

YP 004396914.1 RecD/TraA familv helicase [Clostridium botulinum BKT0159251

YP 002950456, 1 helicase, RecD/TraA family [Geobacillus sp. WCH701 >gb|ACS25190.1 |

YP 387402, 1 UvrD/REP helicase rDesulfovibrio alaskensis G201 >gb|ABB37707.1 |

YP 002771377, 1 hypothetical protein BBR47 18960 rBrevibacillus brevis NBRC 1005991

ZP 01 173819.1 YrrC [Bacillus sp. NRRL B-1491 11 >gb|EAR63466.1 | YrrC [Bacillus sp.

ZP 02074502.1 hypothetical protein CLOL250 01272 [Clostridium sp. L2-501

ZP 02951515.1 helicase, RecD/TraA family [Clostridium butyricum 55211

ZP 07326697.1 helicase, RecD/TraA family [Acetivibrio cellulolvticus CD21

ZP 08662208. 1 helicase, RecD/TraA familv [Streptococcus sp. oral taxon 056 str. F04181

ZP 07709630. 1 helicase, RecD/TraA family protein [Bacillus sp. m3-131

ZP 08151018. 1 RecD/TraA family helicase [Lachnospiraceae bacterium 4 1 37FAA1

ZP 05855961 . 1 helicase, RecD/TraA family [Blautia hansenii DSM 205831

ZP 08333468. 1 RecD/TraA family helicase [Lachnospiraceae bacterium 6 1 63FAA1

ZP 01967327. 1 hypothetical protein RUMTOR 00874 rRuminococcus torques ATCC

ZP 07843612.1 helicase, RecD/TraA familv [Staphylococcus hominis subsp. hominis

ZP 08335292.1 RecD/TraA family helicase [Lachnospiraceae bacterium 9 1 43BFAA1

YP 002425519, 1 helicase, RecD/TraA family [Acidithiobacillus ferrooxidans ATCC

ZP 08074741.1 Exodeoxyribonuclease V [Methyl ocystis sp. ATCC 492421

ZP 03288021.1 hypothetical protein CLONEX 00200 [Clostridium nexile DSM 17871 NP 349457. 1 ATP-dependent exoDNAse (exonuclease V), alpha subunit, RecD YP 535621. 1 exodeoxyribonuclease V alpha chain [Lactobacillus salivarius UCC 1181 YP 001514053.1 RecD/TraA family helicase TAlkaliphilus oremlandii OhlLAsl

ZP 06059658.1 RecD/TraA family helicase Γ Streptococcus sp. 2 1 36FAA1

ZP 04059935. 1 helicase, RecD/TraA family [Staphylococcus hominis SKI 191

AEN87514.1 Exodeoxyribonuclease V-like protein [Bacillus megaterium WSH-0021 YP 003988429.1 helicase, RecD/TraA familv TGeobacillus sp. Y4.1MC11

NP 781026.1 exodeoxyribonuclease V alpha chain [Clostridium tetani E881

YP 004707292.1 hypothetical protein CXIVA 02230 [Clostridium sp. SY85191

YP 003590771.1 helicase, RecD/TraA familv [Bacillus tusciae DSM 29121

ZP 03708405.1 hypothetical protein CLOSTMETH 03166 [Clostridium methylpentosum ZP 07904646.1 RecD/TraA family helicase [Eubacterium saburreum DSM 39861 ZP 08463854.1 exodeoxyribonuclease V alpha subunit [Desmospora sp. 84371

YP 003339261 , 1 exodeoxyribonuclease V [Streptosporangium roseum DSM 430211 ZP 07356412.1 helicase, RecD/TraA family [Desulfovibrio sp. 3 1 syn31

YP 004310482.1 helicase, RecD/TraA family [Clostridium lentocellum DSM 54271 YP 003565054.1 helicase, RecD/TraA family [Bacillus megaterium QM B 15511

EGM50608.1 helicase, RecD/TraA familv [Lactobacillus salivarius GJ-241

ZP 07454758.1 RecD/TraA family helicase [Eubacterium yurii subsp. margaretiae ATCC CBL17987.1 helicase, putative, RecD/TraA family rRuminococcus sp. 18P131 ZP 03917092. 1 possible exodeoxyribonuclease V alpha subunit [Anaerococcus

ZP 08757131. 1 helicase, RecD/TraA family fParvimonas sp. oral taxon 393 str. F04401 EGL99465.1 recD-like DNA helicase YrrC [Lactobacillus salivarius NIAS8401 ZP 01725995. 1 hypothetical protein BB 14905 09550 [Bacillus sp. B149051

YP 003590260.1 helicase. RecD/TraA family [Bacillus tusciae DSM 29121

ZP 07206301.1 helicase, RecD/TraA family [Lactobacillus salivarius ACS-1 16-V-Col5al YP 001680910, 1 exodeoxyribonuclease V. alpha chain. RecD rHeliobacterium

YP 003821341.1 helicase, RecD/TraA family [Clostridium saccharolyticum WM11 ZP 08005791.1 YrrC protein [Bacillus sp. 2 A 57 CT21 >gb IEFV77442. i l YrrC protein YP 002560662.1 exodeoxyribonuclease V alpha subunit [Macrococcus caseolyticus ZP 05028653.1 hypothetical protein MC7420 1174 [Microcoleus chthonoplastes PCC CCC58043.1 RecD-like DNA helicase YrrC [Caloramator australicus RC31

YP 001699482.1 exodeoxyribonuclease V-like protein [Lysinibacillus sphaericus C3-411 ZP 02616886. 1 helicase, RecD/TraA familv [Clostridium botulinum Bfl

XP 001420006.1 predicted protein [Ostreococcus lucimarinus CCE99011

YP 001779796.1 RecD/TraA family helicase [Clostridium botulinum Bl str. Okral YP 001389532.1 RecD/TraA family helicase [Clostridium botulinum F str. Langelandl ZP 02993715. 1 hypothetical protein CLOSPO 00789 [Clostridium sporogenes ATCC ZP 01964108. 1 hypothetical protein RUMOBE 01832 [Ruminococcus obeum ATCC ZP 03227028.1 ATP-dependent exonuclease V [Bacillus coahuilensis m4-41

YP 001252714.1 helicase, RecD/TraA family [Clostridium botulinum A str. ATCC 35021 YP 001307573 , 1 RecD/TraA family helicase [Clostridium beiierinckii NCEVIB 80521 ZP 08091201.1 hypothetical protein FFMPREF9474 02952 [Clostridium symbiosum CBZO 1987.1 recd-like DNA helicase YrrC [Clostridium botulinum H04402 0651 ZP 06620580.1 helicase, RecD/TraA family [Turicibacter sanguinis PC9091

ZP 02612165.1 helicase, RecD/TraA family [Clostridium botulinum NCTC 29161 ZP 03464124.1 hypothetical protein BACPEC 03225 [Bacteroides pectinophilus ATCC ZP 05427870.1 helicase, RecD/TraA family [Eubacterium saphenum ATCC 499891 ZP 04819493. 1 exodeoxyribonuclease V alpha subunit [Staphylococcus epidermidis CBL16176.1 helicase, putative, RecD/TraA familv [Ruminococcus bromii L2-631 CBK73489.1 helicase, putative, RecD/TraA familv [Butyrivibrio fibrisolvens 16/41 XP 003081706.1 Dehydrogenase kinase (ISS) [Ostreococcus tauril >emb|CAL56230.1 | ZP 06425429. 1 helicase, RecD/TraA familv [Peptostreptococcus anaerobius 653 -LI ZP 08539226. 1 helicase, RecD/TraA family [Oribacterium sp. oral taxon 108 str. F04251 ZP 04008608.1 exodeoxyribonuclease V alpha chain [Lactobacillus salivarius ATCC ZP 08525848.1 helicase, RecD/TraA family [Streptococcus anginosus SK521

ZP 08245897.1 helicase, RecD/TraA family [Streptococcus parauberis NCFD 20201 ZP 06290581.1 helicase, RecD/TraA familv [Peptoniphilus lacrimalis 315-B1

ZP 08680798.1 RecD/TraA family helicase [Sporosarcina newyorkensis 26811 451 YP 002802482.1 helicase, RecD/TraA family [Clostridium botulinum A2 str. Kvotol

452 YP 001449573.1 RecD/TraA family helicase Γ Streptococcus gordonii str. Challis substr.

453 ZP 01862085.1 hypothetical protein BSG1 18450 [Bacillus sp. SG-11 >sb|EDL62855.1 |

454 YP 001785497, 1 RecD/TraA family helicase [Clostridium botulinum A3 str. Loch Mareel

455 EFV89168.1 exodeoxvribonuclease V alpha chain [Staphylococcus epidermidis

456 ZP 07956105. 1 RecD/TraA family helicase TLachnospiraceae bacterium 5 1 63FAA1

457 ZP 03055915. 1 helicase, RecD/TraA family [Bacillus pumilus ATCC 70611

458 ZP 04797338. 1 exodeoxvribonuclease V alpha subunit [Staphylococcus epidermidis

459 EGS77340. 1 helicase, RecD/TraA family [Staphylococcus epidermidis VCU1051

460 YP 004478259.1 hypothetical protein STP 0139 [Streptococcus parauberis KCTC 1 15371

461 ZP 08605488.1 RecD/TraA family helicase TLachnospiraceae bacterium

462 ZP 08643227.1 hypothetical protein BRLA c44940 [Brevibacillus laterosporus LMG

463 ZP 06875615.1 putative exonuclease with DNA/RNA helicase motif [Bacillus subtilis

464 ZP 04678546, 1 helicase, RecD/TraA family [Staphylococcus warneri L376031

465 ZP 06613101 , 1 conserved hypothetical protein [Staphylococcus epidermidis

466 ZP 02441 706.1 hypothetical protein ANACOL 00987 [Anaerotruncus colihominis DSM

467 YP 188759.1 RecD/TraA family helicase [Staphylococcus epidermidis RP62A1

468 ZP 02440294. 1 hypothetical protein CLOSS21 02797 [Clostridium sp. SS2/11

469 YP 001919909.1 helicase, RecD/TraA family [Clostridium botulinum E3 str. Alaska E431

470 YP 001884722.1 helicase, RecD/TraA family [Clostridium botulinum B str. Eklund 17B1

471 ZP 02039032.1 hypothetical protein BACCAP 04681 TBacteroides capillosus ATCC

472 ZP 07093704.1 helicase, RecD/TraA family rPeptoniphilus sp. oral taxon 836 str. F01411

473 YP 001487615.1 exodeoxvribonuclease V alpha subunit [Bacillus pumilus SAFR-0321

474 NP 764857. 1 deoxyribonuclease [Staphylococcus epidermidis ATCC 122281

475 YP 804665. 1 ATP-dependent RecD/TraA family DNA helicase [Pediococcus

476 NP 942288.1 exodeoxvribonuclease V alpha chain [Synechocvstis sp. PCC 68031

477 ZP 07054718.1 RecD/TraA family helicase [Listeria gravi DSM 206011 >gb|EFI83599.1 |

478 EHA31059.1 hypothetical protein BSSC8 15020 [Bacillus subtilis subsp. subtilis str.

479 CBL39055.1 helicase. putative. RecD/TraA family rbutvrate-producing bacterium

480 EGF05416. 1 exodeoxvribonuclease V alpha subunit [Streptococcus sanguinis SK10571

481 YP 003974155.1 putative exonuclease [Bacillus atrophaeus 19421 >gb|ADP33224.1 |

482 ZP 07822731 .1 helicase. RecD/TraA family rPeptoniphilus harei ACS-146-V-Sch2bl

483 ZP 06348052.1 helicase, RecD/TraA family [Clostridium sp. M62/11 >gb IEFE10725. i l

484 ZP 05394746.1 helicase, RecD/TraA family [Clostridium carboxidivorans P71

485 YP 004204562.1 putative exonuclease [Bacillus subtilis BSn51 >dbi IB AI86231. i l

486 EGG96535.1 helicase, RecD/TraA family [Staphylococcus epidermidis VCU1211

487 YP 003471 518, 1 Exodeoxvribonuclease V subunit alpha [Staphylococcus lugdunensis

488 ZP 04820712, 1 helicase, RecD/TraA family [Clostridium botulinum El str. 'BoNT E

489 EGF05816.1 exodeoxvribonuclease V alpha subunit [Streptococcus sanguinis SK11

490 YP 002634323.1 hypothetical protein Sea 1231 [Staphylococcus carnosus subsp. carnosus

491 YP 301232, 1 ATP-dependent exonuclease V alpha subunit [Staphylococcus

492 ZP 07841 151. 1 helicase, RecD/TraA family [Staphylococcus caprae C871

493 NP 846841.1 helicase [Bacillus anthracis str. Amesl >ref|YP 021271.21 helicase

494 NP 390625.1 exonuclease with DNA RNA helicase motif [Bacillus subtilis subsp.

495 ZP 07910981.1 RecD/TraA familv helicase [Staphylococcus lugdunensis M235901

496 YP 001727916.1 exonuclease V subunit alpha [Leuconostoc citreum KM201

497 YP 030537. 1 helicase [Bacillus anthracis str. Sternel >ref|ZP 00394720.11 COG0507:

498 ZP 04291295.1 Helicase, RecD/TraA [Bacillus cereus R3098031 >gb|EEK76998.1 |

499 YP 002751754.1 putative helicase [Bacillus cereus 03BB 1021 >gb|AC031219.1 | putative

500 ZP 08091585.1 hypothetical protein HMPREF9474 03336 [Clostridium svmbiosum

The RecD helicase is more preferably one of the helicases shown in Table 5 below or variant thereof. The RecD helicase more preferably comprises the sequence of one of the helicases shown in Table 5, i.e. one of SEQ ID NOs: 18, 21 , 24, 25, 28, 30, 32, 35, 37, 39, 41 and 44, or a variant thereof. Table 5 - More preferred RecD helicases

matii

Deinococc NCBI Reference GGPGTGKS (22) YALTVH

RecD

us Sequence: RGQG

44 2 67

maricopen YP 004170918. (45)

Dma

sis 1

All sequences in the above Table comprise a RecD-like motif V (as shown). Only SEQ ID NOs: 18, 25, 28, 30, 35, 37, 39 and 42 comprise a RecD motif V (as shown).

The RecD helicase is preferably a Tral helicase or a Tral subgroup helicase. Tral helicases and Tral subgroup helicases may contain two RecD helicase domains, a relaxase domain and a C-terminal domain. The Tral subgroup helicase is preferably a TrwC helicase. The Tral helicase or Tral subgroup helicase is preferably one of the helicases shown in Table 6 below or a variant thereof.

The Tral helicase or a Tral subgroup helicase typically comprises a RecD-li ke motif I as defined above (SEQ ID NO : 8) and/or a RecD-like motif V as defined above (SEQ ID NO: 16). Hie Tral helicase or a Tral subgroup helicase preferably compri ses both a RecD-li ke motif I (SEQ ID NO: 8) and a RecD-like motif V (SEQ ID NO: 16). The Tral helicase or a Tral subgroup helicase typically further comprises one of the following two motifs:

- The amino acid motif H-(X l)rX2-R-(X3)s.i2-H-X4-H (hereinafter called the MobF motif HI, SEQ ID NOs: 46 to 53 show all possible MobF motifs ill (including all possible numbers of X3)), wherein XI and X3 are any amino acid and X2 and X4 are independently selected from any amino acid except D, E, K and R. (Xl)₂ is of course Xla-Xlb. Xla and Xlb can be the same of different amino acid. Xla is preferably D or E. X lb is preferably T or D. (Xl)₂ is preferably DT or ED. (Xl}i is most preferably DT. The 5 to 12 amino acids in (X3)₅_i₂ can be the same or different. X2 and X4 are independently selected from G, P, A, V, L, I, M, C, F, Y, W, H, Q. N, S and T. X2 and X4 are preferably not charged. X2 and X4 are preferably not H. X2 is more preferably N, S or A. X2 is most, preferably N. X4 is most preferably F or T. (X3)₅,i2 is preferably 6 or 10 residues in length (SEQ ID NOs: 47 and 5 1 ). Suitable

embodiments of (X3)₃.₁₂ can be derived from SEQ ID NOs: 61 , 65, 69, 73, 74, 82, 86, 90, 94, 98, 102, 1 10, 1 12, 1 13 , 1 14, 1 17, 121 , 124, 125, 129, 133, 136, 140, 144, 147, 151 , 152, 156, 160, 164 and 168shown in Table 7 below (i.e. all but SEQ ID NOs: 78 and 106). Preferred em bodiments of the MobF motif 111 are shown in Table 7 bel ow.

- The amino acid motif G-X1-X2-X3-X4-X5-X6-X7~H-(X8)₆.₁₂-H-X9 (hereinafter called the MobQ motif ill; SEQ ID NOs: 54 to 60 show all possible MobQ motifs ΠΙ (including ail possible numbers of X8)), wherein XI , X2, X3, X5, X6, X? and X9 are independently selected from any amino acid except Γ⁾, E, K and R, X4 is D or E and X8 i s any amino acid. X I, X2, X3, X5, X6, X7 and X9 are independently selected from G, P, A, V, L, i, M, C, F, Y, W, H. Q, N, S and T. XL X2. X3, X5, X6, X7 and X9 are preferably not charged. XI, X2, X3_? X5, X6, X7 and X9 are preferably not H. The 6 to 12 amino acids in (X8 e.{2 can be the same or different. Suitable embodiments of (X8)₆-i;> can be derived from SEQ ID NQs: 78 and 106 shown in Table 7 below. Preferred embodiments of the obF motif III are shown in Table 7 below.

Table 6 - Preferred Tral helicases and Tral subgroup helicases and their Accession Numbers

1 NP 061483.1 conjugal transfer nickase/helicase Tral iPlasmid Fl

2 NP 862951.1 conjugal transfer nickase/helicase Tral [Escherichia coli]

3 ZP 03047597. type IV secretion-like conjugative transfer relaxase protein Tral

4 YP 00203889 conjugal transfer nickase/helicase Tral [Salmonella enterica subsp.

5 YP 001 73989 type IV secretion-like conjugative transfer relaxase protein Tral

6 YP 1901 5, 1 conjugal transfer nickase/helicase Tral [Escherichia coli]

7 ZP 08368984. conjugative transfer relaxase protein Tral [Escherichia coli TA2711

8 EFW76779.1 IncF plasmid conjugative transfer DNA-nicking and unwinding

9 YP 00382905 type IV secretion-like conjugative transfer relaxase protein

10 EGX 11991. 1 conjugative transfer relaxase protein Tral [Escherichia coli

11 YP 00191916 type IV secretion-like conjugative transfer relaxase protein Tral

12 ZP 03051102. type IV secretion-like conjugative transfer relaxase protein Tral

13 EGH36328. 1 IncF plasmid conjugative transfer DNA-nicking and unwinding

14 ZP 03030171. type IV secretion-like conjugative transfer relaxase protein Tral

15 EGB 88794.1 conjugative transfer relaxase protein Tral [Escherichia coli MS 117-31

16 YP 00382916 nickase/helicase [Escherichia colil >gb|ADL14054.1 | Tral

17 EFU55615.1 conjugative transfer relaxase protein Tral [Escherichia coli MS 16-31

18 EGB69775. 1 conjugative transfer relaxase Tral [Escherichia coli TW105091

19 YP 00303405 conjugal transfer nickase/helicase Tral [Escherichia coli Vir681

20 YP 443956.1 conjugal transfer nickase/helicase Tral [Escherichia coli]

21 YP 00196541 oriT-specific relaxase; helicase [Escherichia colil >gb|ABG29544.1 |

22 AA098619.1 DNA helicase I [Escherichia colil

23 YP 00240109 conjugal transfer nickase/helicase Tral [Escherichia coli S881

24 YP 00233218 conjugal transfer nickase/helicase Tral [Escherichia coli 0127:H6 str.

25 CBG27820.1 DNA helicase I [Escherichia colil

26 YP 00171193 conjugal transfer nickase/helicase Tral [Escherichia colil

27 EGI88721.1 conjugative transfer relaxase protein Tral [Shigella dysenteriae 155-

28 ZP 06661276. conjugative transfer relaxase Tral [Escherichia coli B0881

29 YP 00148121 conjugal transfer nickase/helicase Tral [Escherichia coli APEC Oil

30 ZP 03070008. type IV secretion-like conjugative transfer relaxase protein Tral

31 YP 00191934 type IV secretion-like conjugative transfer relaxase protein Tral

32 YP 00129475 conjugal transfer nickase/helicase Tral [Escherichia colil

33 YP 00323254 conjugal transfer protein Tral [Escherichia coli 026:H11 str. 113681

34 YP 00323781 nickase [Escherichia coli 0111 :H- str. 11 1281 >dbi IBAI39380. i l

35 ADR29948.1 conjugative transfer relaxase protein Tral [Escherichia coli 083 :H1

36 YP 00181654 conjugal transfer nickase/helicase Tral [Escherichia coli 15201

37 ZP 07104698. conjugative transfer relaxase protein Tral [Escherichia coli MS 119-71

38 ZP 06988741. conjugal transfer nickase/helicase Tral [Escherichia coli FVEC13021

39 YP 00322510 putative Tral protein [Escherichia coli O103 :H2 str. 120091

40 AEE59988.1 IncF transfer nickase/helicase protein Tral [Escherichia coli

41 EFZ76933.1 conjugative transfer relaxase protein Tral [Escherichia coli RN587/11

42 YP 00487008 protein Tral [Escherichia colil >gb|AEP03777.1 | Tral [Escherichia

43 ZP 08376536. conjugative transfer relaxase protein Tral [Escherichia coli H5911

44 YP 538737.1 DNA helicase I [Escherichia coli UTI891 >ref|ZP 030351 19.11 type

45 NP 052981.1 conjugal transfer nickase/helicase Tral [Plasmid R1001

46 YP 00329403 conjugal transfer nickase/helicase [Escherichia coli ETEC HI 04071

47 YP 00240597 conjugal transfer protein Tral [Escherichia coli UMN0261

48 ZP 08344394. conjugative transfer relaxase protein Tral [Escherichia coli H7361

49 ZP 06648569. conjugal transfer nickase/helicase Tral [Escherichia coli FVEC14121

50 CBJ04377.1 DNA helicase I (Tral) (EC 3.6.1.-) [Escherichia coli ETEC H104071

51 YP 00332919 Tral [Klebsiella pneumoniael >gb|ACK98846.1 | Tral [Klebsiella

52 ADN74088.1 conjugal transfer nickase/helicase Tral [Escherichia coli UM1461

53 YP 00351762 Tral [Klebsiella pneumoniael >gb|ADD63581.1 | Tral [Klebsiella

54 YP 788091 , 1 conjugal transfer nickase/helicase Tral [Escherichia colil

55 P22706.1 RecName: Full=Multifunctional conjugation protein Tral; Includes:

56 EGC09761.1 conjugative transfer relaxase Tral [Escherichia coli El 1671

57 EGX11419. 1 conjugative transfer relaxase protein Tral [Escherichia coli

58 YP 00393764 protein Tral (DNA helicase I) [Escherichia colil >emb I CBX35963. i l EGW83369.1 conjugative transfer relaxase protein Tral [Escherichia coli EGB59895.1 conjugative transfer relaxase Tral [Escherichia coli M8631 EFZ69441.1 conjugative transfer relaxase protein Tral [Escherichia coli EFU44479, 1 conjugative transfer relaxase protein Tral [Escherichia coli MS YP 406350.1 oriT nicking and unwinding protein, fragment [Shigella boydii NP 085415.1 oriT nicking and unwinding protein, fragment [Shigella flexneri EGW99614.1 conjugative transfer relaxase protein Tral [Escherichia coli EG 37046.1 conjugative transfer relaxase protein Tral [Shigella flexneri K- EFW 60434, 1 IncF plasmid conjugative transfer DNA-nicking and unwinding ZP 07678376.1 conjugative transfer relaxase protein Tral [Shigella dysenteriae EFW49146.1 IncF plasmid conjugative transfer DNA-nicking and unwinding AEG39580.1 IncF plasmid conjugative transfer DNA-nicking and unwinding NP 490592.1 conjugal transfer nickase/helicase Tral [Salmonella tvphimurium EFW56310.1 IncF plasmid conjugative transfer DNA-nicking and unwinding YP 271768.1 conjugal transfer nickase/helicase Tral [Salmonella enterical EGP21913.1 Protein tral [Escherichia coli PCN0331

EGR7091 1.1 conjugal transfer nickase/helicase Tral [Escherichia coli EGB84217.1 conjugative transfer relaxase protein Tral [Escherichia coli MS ZP 07119795.1 conjugative transfer relaxase protein Tral [Escherichia coli MS YP 313447.1 oriT nicking and unwinding protein, fragment [Shigella sonnei EFU49447.1 conjugative transfer relaxase protein Tral [Escherichia coli MS ZP 07 97893.1 conjugative transfer relaxase protein Tral [Escherichia coli MS ZP 08386420.1 conjugative transfer relaxase protein Tral [Escherichia coli ZP 07246816.1 conjugative transfer relaxase protein Tral [Escherichia coli MS YP 406123.1 oriT nicking and unwinding protein, fragment [Shigella

EFZ55097.1 conjugative transfer relaxase protein Tral [Shigella sonnei 53G1 YP 002213911.1 conjugative transfer relaxase protein Tral [Salmonella enterica YP 001716148.1 conjugative transfer oriT nicking-unwinding protein [Salmonella EGE32684. 1 conjugative transfer oriT nicking-unwinding protein [Salmonella EFZ60917.1 conjugative transfer relaxase protein Tral [Escherichia coli LT- EGB40000.1 conjugative transfer relaxase Tral [Escherichia coli H1201 CAH64717.1 putative DNA helicase I [uncultured bacteriuml

ZP 08351681.1 conjugative transfer relaxase protein Tral [Escherichia coli ZP 08351622.1 conjugative transfer relaxase protein Tral [Escherichia coli YP 001338645.1 conjugal transfer nickase/helicase Tral [Klebsiella pneumoniae EGK29 1 1.1 conjugative transfer relaxase protein Tral [Shigella flexneri K- NP 858382.1 oriT nicking and unwinding protein [Shigella flexneri 2a str. EGT71209.1 hypothetical protein C2271 1 5245 [Escherichia coli Ο104Ή4 YP 003560496.1 oriT nicking-unwinding [Klebsiella pneumoniae]

YP 003517517.1 Tral [Klebsiella pneumoniael >reflYP 004249929.11 IncF ZP 06015312.1 conjugal transfer nickase/helicase Tral [Klebsiella pneumoniae EGJ92351.1 conjugative transfer relaxase protein Tral [Shigella flexneri K- YP 003754133.1 conjugal transfer nickase/helicase Tral [Klebsiella pneumoniael ADA76996.1 OriT nicking and unwinding protein [Shigella flexneri 20020171 YP 001 154759.1 conjugal transfer nickase/helicase Tral [Yersinia pestis Pestoides YP 093987.1 conjugal transfer nickase/helicase Tral [Yersinia pestisl

EGB74535. 1 conjugative transfer relaxase protein Tral [Escherichia coli MS EGX15096.1 protein tral domain protein [Escherichia coli TX19991

ZP 07778521.1 tral domain protein [Escherichia coli 2362-751 >gb|EFRl 8955. I I EGB44795.1 DNA helicase Tral [Escherichia coli H2521

ZP 07192950.1 putative conjugative transfer relaxase protein Tral [Escherichia ZP 07692602.1 putative conjugative transfer relaxase protein Tral [Escherichia ZP 07212721.1 putative conjugative transfer relaxase protein Tral [Escherichia ZP 07122964.1 putative conjugative transfer relaxase protein Tral [Escherichia ZP 06641688.1 conjugal transfer nickase/helicase Tral [Serratia odorifera DSM ZP 07213 1 12. 1 putative conjugative transfer relaxase protein Tral [Escherichia ZP 07125330. ] putative conjugative transfer relaxase protein Tral [Escherichia AAA98086.1 helicase I TPlasmid Fl >gb|AAC44187.1 | Tral* [Escherichia ZP 07692570, 1 DNA helicase Tral [Escherichia coli MS 145-71 EGB44794.1 conjugative relaxase domain-containing protein rEscherichia coli

CBA76609.1 conjugal transfer nickase/helicase TArsenophonus nasoniael

YP 004831100.1 conjugal transfer nickase/helicase Tral TSerratia marcescensl

AEJ60155.1 conjugal transfer protein Tral [Escherichia coli UMNF 181

EGX24402.1 conjugative transfer relaxase protein Tral [Escherichia coli

AAM90727.1 Tral [Salmonella enterica subsp. enterica serovar Typhil

ZP 08374850.1 conjugative transfer relaxase protein Tral [Escherichia coli

CBY99022.1 conjugal transfer nickase/helicase Tral [Salmonella enterica

ZP 02347591 . 1 conjugative transfer relaxase protein Tral [Salmonella enterica

YP 001144265.1 Tral protein [Aeromonas salmonicida subsp. salmonicida A4491

YP 001144345.1 Tral protein [Aeromonas salmonicida subsp. salmonicida A4491

YP 003717559.1 putative Tral DNA helicase I [Escherichia coli ETEC 1392/751

ZP 08351623.1 protein Tral (DNA helicase I) [Escherichia coli M6051

ZP 06658914.1 conjugative transfer relaxase Tral [Escherichia coli B 1851

YP 002291232, 1 Tral protein [Escherichia coli SE111 >dbj |BAG80410.1 | Tral

EFZ47384.1 protein tral domain protein [Escherichia coli E1280101

EGX19478.1 protein tral domain protein [Escherichia coli STEC SI 1911

YP 002527579.1 hypothetical protein pO103 123 [Escherichia colil

EGX15Q95.1 protein tral domain protein [Escherichia coli TX19991

EFZ47387.1 protein tral domain protein [Escherichia coli E1280101

ZP 06193648. 1 protein Tral [Serratia odorifera 4Rxl31 >gb IEFA13747. i l protein

EGB39999.1 conjugative relaxase domain-containing protein [Escherichia coli

YP 003739388.1 conjugal transfer nickase/helicase [Erwinia billingiae Eb6611

YP 002539341.1 Tral [Escherichia colil >gb|ACM18376.1 | Tral [Escherichia colil

BAA31818.1 helicase I [Escherichia coli 0157:H7 str. Sakail

ADA76995.1 OriT nicking and unwinding protein [Shigella flexneri 20020171

EFZ45075.1 protein tral domain protein [Escherichia coli E1280101

ZP 05940744.1 conjugal transfer protein Tral [Escherichia coli 0157:H7 str.

NP 858381.1 oriT nicking and unwinding protein [Shigella flexneri 2a str. 3011

YP 325658.1 DNA helicase [Escherichia coli 0157:H7 EDL9331

EFZ04572.1 Tral protein [Salmonella enterica subsp. enterica serovar

YP 209287.1 Tral protein [Salmonella enterica subsp. enterica serovar

YP 001598090.1 hypothetical protein pOU7519 37 [Salmonella enterica subsp.

EGY27955.1 DNA helicase [Candidatus Regiella insecticola R5.151

ZP 02775703.2 protein Tral [Escherichia coli 0157:H7 str. EC41131

ZP 02802536.2 protein Tral (DNA helicase I) [Escherichia coli 0157:H7 str.

ZP 07779988.1 tral domain protein [Escherichia coli 2362-751 >gb|EFR17488.1 |

ZP 08386421.1 protein Tral (DNA helicase I) [Escherichia coli H2991

1P4D A Chain A, F Factor Trai Relaxase Domain >pdb| lP4D|B Chain B,

2A0I A Chain A, F Factor Trai Relaxase Domain Bound To F Orit

EFZ47389.1 protein tral domain protein [Escherichia coli E1280101

YP 004118615.1 conjugative transfer relaxase protein Tral [Pantoea sp. At-9bl

YP 004119632.1 conjugative transfer relaxase protein Tral [Pantoea sp. At-9bl

EGW99739.1 protein tral domain protein [Escherichia coli G58-11

YP 004821631.1 conjugative transfer relaxase protein Tral [Enterobacter asburiae

YP 001165588.1 exonuclease V subunit alpha [Enterobacter sp. 6381

YP 003602677.1 conjugative transfer relaxase protein Tral [Enterobacter cloacae

YP 31 1531. 1 hypothetical protein SSON 2674 [Shigella sonnei Ss0461

NP 073254. 1 hvpothetical protein pKDSC50 p30 [Salmonella enterica subsp.

2Q7T A Chain A, Crystal Structure Of The F Plasmid Trai Relaxase

EGB84260.1 conjugative relaxase domain protein [Escherichia coli MS 60-11

ZP 07119794. 1 conjugative relaxase domain protein [Escherichia coli MS 198-11

EGB74274.1 conjugative relaxase domain protein [Escherichia coli MS 57-21

ZP 04533197. 1 helicase I [Escherichia sp. 3 2 53FAA1 >gb|EEH89372.1 |

EFU49424. 1 conjugative relaxase domain protein [Escherichia coli MS 153-11

EFZ60914.1 protein tral domain protein [Escherichia coli LT-681

CBK86956.1 conjugative relaxase domain, TrwC/Tral family [Enterobacter

YP 313450, 1 oriT nicking and unwinding protein, fragment [Shigella sonnei

EGP22056.1 hypothetical protein PPECC33 45560 [Escherichia coli PCN0331 ZP 04533202. 1 Tral protein rEscherichia sp. 3 2 53FAA1 >gb|EEH89366.1 | Tral

ZP 07248320. 1 DNA helicase Tral rEscherichia coli MS 146-11 >gb|EFK88152.1 |

EFZ45103.1 protein tral domain protein rEscherichia coli E1280101

YP 003502675 , 1 ATP-dependent exoDNAse (exonuclease V), alpha subunit -

ZP 04533172.1 predicted protein rEscherichia sp. 3 2 53FAA1 >gb|EEH89397.1 |

YP 406124. 1 putative DNA helicase I, fragment [Shigella dysenteriae Sdl971

ZP 04533171.1 conserved hypothetical protein rEscherichia sp. 3 2 53FAA1

ZP 0719295 1 .1 DNA helicase Tral rEscherichia coli MS 196-11 >gb|EFI85454.1 |

YP 001853797.1 putative coniugative transfer protein Tral rVibrio tapetisl

YP 00226151 1 .1 protein Tral (DNA helicase I) rAliivibrio salmonicida LFI12381

NP 762615.1 coniugative transfer relaxase protein Tral TVibrio vulnificus

YP 001393155.1 putative coniugative transfer protein Tral TVibrio vulnificusl

EGB74536.1 DNA helicase Tral rEscherichia coli MS 57-21

NP 932226.1 putative coniugative transfer protein Tral rVibrio vulnificus

YP 001557030, 1 coniugative transfer relaxase protein Tral iShewanella baltica

ADT96679.1 coniugative transfer relaxase protein Tral iShewanella baltica

YP 00191 1094.1 Tral protein rErwinia tasmaniensis Et 1/991 >emb|CA094972.11

YP 002360275.1 coniugative transfer relaxase protein Tral iShewanella baltica

AEG13610.1 coniugative transfer relaxase protein Tral iShewanella baltica

EFZ04571.1 Tral protein rSalmonella enterica subsp. enterica serovar

ZP 01813760. 1 putative coniugative transfer protein Tral iVibrionales bacterium

YP 002360333.1 coniugative transfer relaxase protein Tral iShewanella baltica

YP 001557007.1 coniugative transfer relaxase protein Tral iShewanella baltica

YP 002364244.1 coniugative transfer relaxase protein Tral iShewanella baltica

YP 209286, 1 Tral protein iSalmonella enterica subsp. enterica serovar

YP 015476, 1 DNA helicase Tral iPhotobacterium profundum SS91

YP 001355447.1 coniugative transfer relaxase protein Tral iShewanella baltica

EHC04201.1 coniugative transfer relaxase protein Tral iShewanella baltica

ZP 06188936.1 coniugative transfer relaxase protein Tral iLegionella

ZP 06157867.1 IncF plasmid coniugative transfer DNA-ni eking and unwinding

ZP 06157920. 1 IncF plasmid coniugative transfer DNA-ni eking and unwinding

ZP 08351743. 1 protein Tral (DNA helicase I) iEscherichia coli M6051

ZP 08738328. 1 putative coniugative transfer protein Tral TVibrio tubiashii ATCC

YP 003993727.1 incf plasmid coniugative transfer DNA-nicking and unwinding

ZP 06157811.1 IncF plasmid coniugative transfer DNA-nicking and unwinding

YP 00391 5110.1 putative coniugative transfer protein Tral TLegionella

YP 122194. 1 hvpothetical protein plpp0039 TLegionella pneumophila str. Parisl

ZP 07197892.1 coniugative relaxase domain protein TEscherichia coli MS 185-11

ZP 05884791.1 putative coniugative transfer protein Tral TVibrio coralliilyticus

ZP 07222592.1 type-F coniugative transfer system pilin acetylase TraX

3FLD A Chain A, Crystal Structure Of The Trai C-Terminal Domain

EGT71207.1 hypothetical protein C22711 5243 TEscherichia coli O 104:H4 str.

ZP 05440093. 1 coniugal transfer nickase/helicase Tral TEscherichia sp. 4 1 40B1

EGX24451.1 protein tral domain protein TEscherichia coli TX19991

EFZ55098.1 tral domain protein TShigella sonnei 53G1

YP 003933505.1 DNA methylase TPantoea vagans C9-H >gb|ADO08159.1 |

AAA83930.1 tral TPlasmid Fl

ADQ53972.1 putative coniugative transfer protein TVibrio harveyil

ZP 07778522. 1 tral domain protein TEscherichia coli 2362-751 >gb|EFR18956.1 |

ZP 07192561.1 conserved domain protein TEscherichia coli MS 196-11

AAW64824.1 oriT nicking and unwinding protein TShigella flexneril

NP 085414.1 oriT nicking and unwinding protein, fragment TShigella flexneri

ADA76994.1 OriT nicking and unwinding protein TShigella flexneri 20020171

ZP 07197891. 1 coniugative relaxase domain protein TEscherichia coli MS 185-11

EFU44520. 1 coniugative relaxase domain protein TEscherichia coli MS 1 10-31

ZP 07222591.1 coniugative relaxase domain protein TEscherichia coli MS 78-11

YP 406349, 1 oriT nicking and unwinding protein, fragment TShigella boydii

YP 406122, 1 oriT nicking and unwinding protein, fragment TShigella

EFW49145.1 coniugal transfer nickase/helicase Tral TShigella dysenteriae CDC ZP 07246817. 1 conjugative relaxase domain protein [Escherichia coli MS 146-11

YP 004250852.1 putative protein tral (DNA helicase I) TVibrio nigripulchritudol

EFZ60915.1 protein tral domain protein [Escherichia coli LT-681

YP 617529. 1 TrwC protein rSphingopyxis alaskensis RB22561

ZP 01813650.1 ATP-dependent exoDNAse, alpha subunit TVibrionales bacterium

ZP 01813651.1 ATP-dependent exoDNAse, alpha subunit [Vibrionales bacterium

NP 052850.1 hvpothetical protein QpDV p09 TCoxiella burnetiil

CAA75825.1 hvpothetical protein TCoxiella burnetiil

YP 002302593.1 DNA helicase TCoxiella burnetii CbuK 01541 >gb|ACJ21266.1 |

YP 003502676.1 Tral [Escherichia coli 055 :H7 str. CB96151 >gb|ADD59692.1 |

YP 001649308.1 putative protein tral TCoxiella burnetii 'MSU Goat Q177'l

YP 001423428,2 DNA helicase TCoxiella burnetii Dugway 5J108-1 1 11

YP 001595803.1 putative protein tral TCoxiella burnetii RSA 3311

NP 052342. 1 hvpothetical protein QpHl plO TCoxiella burnetiil

ZP 01863208.1 hvpothetical protein ED21 17597 [Erythrobacter sp. SD-211

ZP 08645753. 1 conjugal transfer protein TraA TAcetobacter tropicalis NBRC

YP 497456.1 TrwC protein rNovosphingobium aromaticivorans DSM 124441

AAL78346.1 DNA helicase I [Escherichia colil

EFW60435.1 conjugal transfer nickase/helicase Tral [Shigella flexneri CDC

YP 001235537, 1 exonuclease V subunit alpha TAcidiphilium crvptum JF-51

ZP 05038212.1 hvpothetical protein S7335 4654 iSynechococcus sp. PCC 73351

ZP 08897263.1 exonuclease V subunit alpha TGluconacetobacter oboediens

NP 049139.1 DNA helicase rNovosphingobium aromaticivoransl

YP 004390567.1 conjugative relaxase domain-containing protein TAlicycliphilus

ZP 07678069. 1 TrwC protein TRalstonia sp. 5 7 47FAA1 >refjZP 08896172.1

YP 974028.1 TrwC protein TAcidovorax sp. JS421 >gb|ABM44293.1 | TrwC

YP 001869867.1 mobilization protein Tral-like protein HNostoc punctiforme PCC

YP 718086, 1 DNA helicase TSphingomonas sp. KA11 >dbi IBAF03374. i l DNA

YP 004534199, 1 TrwC protein rNovosphingobium sp. PP1Y1 >emb I CCA92381. i l

ZP 08701842.1 TrwC protein [Citromicrobium sp. JLT13631

YP 003602886.1 hvpothetical protein ECL B l 16 TEnterobacter cloacae subsp.

ZP 06861556.1 TrwC protein TCitromicrobium bathvomarinum JL3541

NP 542915.1 putative TraC protein TPseudomonas putidal >emb|CAC86855.1 |

YP 457045.1 TrwC protein TErvthrobacter litoralis HTCC25941

YP 122325.1 hvpothetical protein plpl0032 [Legionella pneumophila str. Lensl

YP 004030608.1 DNA helicase I Tral TBurkholderia rhizoxinica FOCI 4541

YP 457732, 1 TrwC protein TErvthrobacter litoralis HTCC25941

ZP 01039301.1 TrwC protein TErvthrobacter sp. NAPll >gb|EAQ29772.1 TrwC

YP 737083. 1 TrwC protein TShewanella sp. MR-71 >gb IABI42026. i l TrwC

ZP 05040239.1 hvpothetical protein S7335 1207 rSynechococcus sp. PCC 73351

AA P57243.1 putative TraC protein [Pseudomonas putidal

NP 942625.1 TrwC rXanthomonas citril >reflZP 06705283.11 Tral protein

10MH A Chain A, Conjugative Relaxase Trwc In Complex With Orit Dna.

ZP 06485934.1 TrwC protein TXanthomonas campestris pv. vasculorum

10SB A Chain A, Conjugative Relaxase Trwc In Complex With Orit Dna.

ZP 08207479.1 conjugative relaxase region-like protein rNovosphingobium

2CDM A Chain A, The Structure Of Trwc Complexed With A 27-Mer Dna

ZP 01731779.1 hvpothetical protein CY0110 01035 TCvanothece sp. CCY01101

NP 644759.1 TrwC protein TXanthomonas axonopodis pv. citri str. 3061

ZP 06732867. 1 Tral protein TXanthomonas fuscans subsp. aurantifolii str. ICPB

YP 361538.1 putative Tral protein TXanthomonas campestris pv. vesicatoria str.

CB. 06129.1 putative traC, type IV secretion system TRalstonia solanacearum

YP 001260099, 1 conjugative relaxase region-like protein TSphingomonas wittichii

YP 0014516 1 , 1 putative type IV conjugative transfer system coupling protein

ZP 08208941.1 conjugative relaxase region-like protein rNovosphingobium

AA084912.1 DNA helicase I [Escherichia colil

YP 002515847.1 DNA relaxase/conjugal transfer nickase-helicase TrwC

YP 001260037.1 conjugative relaxase region-like protein TSphingomonas wittichii

ZP 04629294. 1 hvpothetical protein ybercOOOl 36240 TYersinia bercovieri ATCC CAZ 15897.1 probable conjugal transfer protein TXanthomonas albilineansl YP 315578.1 TrwC protein I hiobacillus denitrificans ATCC 252591 ZP 01770026.1 TrwC protein TBurkholderia pseudomallei 3051

YP 001869963.1 exonuclease V subunit alpha TNostoc punctiforme PCC 731021 ZP 08210392.1 TrwC protein rNovosphinsobium nitrogenifigens DSM 193701 YP 001692976, 1 mobilization protein Trai [Yersinia enterocolitical

YP 003455306, 1 conjugative transfer protein Trai [Legionella longbeachae YP 745335. 1 trai protein (DNA helicase I) [Granulibacter bethesdensis YP 00191 1166.1 TrwC [Salmonella enterica subsp. enterica serovar Dublinl YP 001874877.1 mobilisation protein [Providencia rettgeril >emb|CAQ48354.1 | YP 001552064.1 trwC protein [Salmonella enterica subsp. enterica serovar CAA44853.2 TrwC [Escherichia coli K-121

YP 096090.1 hypothetical protein lpg2077 [Legionella pneumophila subsp. YP 534815.1 putative plasmid transfer protein TraC [Pseudomonas putidal FAA00039.1 TPA: TrwC protein [Escherichia colil

NP 863125.1 putative TraC protein [Pseudomonas putidal

ZP 04868849. 1 conserved hypothetical protein [Staphylococcus aureus subsp. ZP 01304707.1 TrwC protein [Sphingomonas sp. SKA581 >gb IEAT07464. i l ZP 05040124.1 TrwC relaxase family [Synechococcus sp. PCC 73351

YP 001798665 , 1 putative TrwC/Tral protein [Cyanothece sp. ATCC 511421 YP 002235496, 1 putative conjugative transfer protein [Burkholderia

CAZ 15872.1 probable mobilization protein trai [Xanthomonas albilineansl YP 840564. 1 TrwC protein [Burkholderia cenocepacia HI24241

ZP 08207332.1 TrwC protein [Novosphingobium nitrogenifigens DSM 193701 YP 001966297.1 Trai [Pseudomonas sp. CT141 >gb|ABA25997.1 | Trai

3L6T A Chain A, Crystal Structure Of An N-Terminal Mutant Of The YP 001736290.1 DNA helicase. TrwC and Trai like protein [Synechococcus sp. 3L57 A Chain A, Crystal Structure Of The Plasmid Pcul Trai Relaxase ZP 04532999. 1 F pilin acetylation protein [Escherichia sp. 3 2 53FAA1 CAA40677.1 DNA helicase I [Escherichia colil

NP 478459.1 hypothetical protein alr8034 [Nostoc sp. PCC 71201

YP 001893556.1 conjugative relaxase domain protein [Burkholderia

YP 001033863.1 hypothetical protein RSP 3904 [Rhodobacter sphaeroides ZP 06064648.1 TrwC protein [Acinetobacter johnsonii SH0461

YP 003829308, 1 nickase/helicase [Escherichia colil >gb|ADL 14202.11 Trai YP 002332893.1 conjugal transfer protein [Klebsiella pneumoniael

YP 002286896, 1 Trai [Klebsiella pneumoniael >gb ACI63157. i l Trai

YP 003813077.1 Trai [Klebsiella pneumoniael >gb IADG84846. i l Trai

YP 002286953.1 Trai [Klebsiella pneumoniael >ref|YP 003675776.11 Trai YP 001096334.1 hypothetical protein pLEW517 p09 [Escherichia colil YP 724504.1 hypothetical protein pMUR050 047 [Escherichia colil NP 51 201.1 hypothetical protein R46 023 [IncN plasmid R461

ADH30046.1 conjugal transfer protein [Escherichia coli 025b:H4-ST131 str. YP 002913254.1 TrwC protein [Burkholderia glumae BGR11 >gb|ACR32934.1 | YP 004362462.1 TrwC protein [Burkholderia gladioli BSR31 >gb|AEA65432.1 | ZP 02468056. 1 TrwC protein [Burkholderia thailandensis MSMB431

YP 001840913.1 TrwC protein [Acinetobacter baumannii ACICUl

ZP 07239267.1 TrwC protein [Acinetobacter baumannii AB0591

YP 003853339, 1 TrwC protein [Parvularcula bermudensis HTCC25031

ZP 07237891.1 TrwC protein [Acinetobacter baumannii AB0581

YP 002491522, 1 conjugative relaxase domain-containing protein

YP 002907678.1 TrwC protein [Burkholderia glumae BGR11 >gb|ACR32827.1 | YP 004350971.1 TrwC protein [Burkholderia gladioli BSR31 >gb|AEA65648.1 | ZP 02834825.2 protein TraD [Salmonella enterica subsp. enterica serovar ADX05370.1 TrwC protein [Acinetobacter baumannii 1656-21

YP 003552078.1 TrwC protein [Candidatus Punicei spirillum marinum

EGB59894.1 trai protein [Escherichia coli M8631

YP 001522461 .1 hypothetical protein AMI F0157 [Acaryochloris marina ZP 05738733. 1 protein Trai [Silicibacter sp. TrichCH4Bl >gb|EEW61008.1 | AEM77047.1 putative conjugative relaxase [Escherichia colil

EHC71302.1 IncW plasmid conjugative relaxase protein TrwC [Salmonella

YP 004765041 , 1 Tral [Escherichia colil >gb|AEK64833.1 | Tral [Escherichia colil YP 004553102, 1 conjugative relaxase domain-containing protein TSphingobium NP 073253. 1 hypothetical protein pKDSC50 p29 [Salmonella enterica subsp. YP 004535774.1 DNA relaxase/conjugal transfer nickase-helicase TrwC

AEA76430.1 VirD2 [Klebsiella pneumoniae]

YP 001806422.1 putative TrwC/Tral protein [Cyanothece sp. ATCC 511421 ΖΡ 08138981. 1 TrwC protein TPseudomonas sp. TJI-511 >gb|EGB99721.1 | TrwC YP 394134.1 exonuclease V subunit alpha TSulfurimonas denitrificans DSM EG061 142.1 conjugative relaxase domain protein [Acidithiobacillus sp. GGI- ΖΡ 06732944.1 Tral protein [Xanthomonas fuscans subsp. aurantifolii str. ICPB YP 004218965.1 conjugative relaxase domain protein [Acidobacterium sp.

YP 001941994.1 relaxase [Burkholderia multivorans ATCC 176161

EDZ39520.1 Protein of unknown function [Leptospirillum sp. Group II '5 -way YP 004415459.1 TrwC protein rPusillimonas sp. T7-71 >gb|AEC18835.1 | TrwC YP 003545248.1 tral/trwC-like protein rSphingobium japonicum UT26S1

YP 004184501.1 conjugative relaxase domain-containing protein TTerriglobus EGD06685.1 relaxase [Burkholderia sp. TJI491

ADQ53945.1 putative conjugative transfer protein [Vibrio harveyil

YP 004089509, 1 conjugative relaxase domain protein f Asticcacaulis excentricus YP 004183694.1 conjugative relaxase domain-containing protein TTerriglobus YP 003900289.1 conjugative relaxase domain-containing protein [Cyanothece sp. EDZ40407.1 Putative mobilization protein TraA [Leptospirillum sp. Group II YP 004534918.1 TrwC protein TNovosphingobium sp. PP1Y1 >emb I CCA93100. i l YP 004210530.1 conjugative relaxase domain protein [Acidobacterium sp.

YP 003642130.1 conjugative relaxase domain protein [Thiomonas intermedia K121 YP 004277247.1 putative relaxase TrwC [Acidiphilium multivorum AIU3011 NP 857772. 1 DNA helicase I [Yersinia pestis KIMl >gb|AAC62598.1 | DNA EGB 74534, 1 hypothetical protein HMPREF9532 05052 [Escherichia coli MS ZP 08138968.1 putative TraC protein TPseudomonas sp. TJI-511

YP 002756187.1 conjugative relaxase domain protein [Acidobacterium capsulatum ZP 07392869. 1 conjugative relaxase domain protein [Shewanella baltica OS1831 YP 004210680.1 conjugative relaxase domain protein [Acidobacterium sp.

EAY56629.1 probable TrwC protein [Leptospirillum rubaruml

YP 068423.1 hypothetical protein pYVOOlO [Yersinia pseudotuberculosis IP NP 995413 , 1 hypothetical protein YP pCD97 [Yersinia pestis biovar Microtus ZP 08634947.1 Conjugative relaxase domain protein [Acidiphilium sp. PM1 ZP 01301850.1 hypothetical protein SKA58 02210 TSphingomonas sp. SKA581 YP 002754293.1 conjugative relaxase domain protein [Acidobacterium capsulatum YP 001818827.1 conjugative relaxase domain-containing protein [Opitutus terrae EGT71208.1 hypothetical protein C22711 5244 [Escherichia coli O 104:H4 str. YP 001522273.1 hypothetical protein AMI E0190 [Acaryochloris marina

YP 003891048.1 conjugative relaxase domain protein [Cyanothece sp. PCC 78221 YP 001521 867, 1 hypothetical protein AMI D0057 [Acaryochloris marina YP 004748378.1 Tral protein [Acidithiobacillus caldus SM-H >gb|AEK57678.1 | YP 002380579.1 relaxase [Cyanothece sp. PCC 74241 >gb|ACK74122.1

ZP 08634902.1 Conjugative relaxase domain protein [Acidiphilium sp. PM1 ZP 05738878. 1 Tral [Silicibacter sp. TrichCH4Bl >gb|EEW61 153.11 Tral YP 001821352.1 conjugative relaxase domain-containing protein [Opitutus terrae ZP 02733385. 1 TrwC protein [Gemmata obscuri globus UQM 22461

YP 004183160.1 conjugative relaxase domain-containing protein [Terriglobus YP 001522155 , 1 TrwC protein, putative [Acaryochloris marina MBIC 110171 YP 002478348, 1 conjugative relaxase domain protein [Cyanothece sp. PCC 74251 YP 002756241.1 conjugative relaxase domain protein [Acidobacterium capsulatum YP 001521036.1 hypothetical protein AMI A0387 [Acaryochloris marina YP 004416953.1 TrwC protein [Pusillimonas sp. T7-71 >gb|AEC20329.1 | TrwC YP 001521806.1 hypothetical protein AMI C0379 [Acaryochloris marina

YP 001357151 .1 hypothetical protein S 1688 rNitratiruptor sp. SB155-21 ZP 07030639. 1 conjugative relaxase domain protein rAcidobacterium sp.

YP 530542.1 putative ATP-dependent exoDNAse (exonuclease V) subunit

NP 052442, 1 hvpothetical protein pYVe227 p65 [Yersinia enterocolitical

YP 004783051 , 1 conjugative relaxase domain-containing protein rAcidithiobaciUus

YP 003262832.1 relaxase [Halothiobacillus neapolitanus c21 >gb|ACX95785.1 |

AD053973.1 putative conjugative transfer protein iVibrio harvevil

I - 13/ 7984. 1 Conjugal transfer protein, TraA rLeptospirillum sp. Group II '5-

YP 001522591.1 hypothetical protein AMI G0097 rAcaryochloris marina

EAY56417.1 putative conjugal transfer protein (TraA) rLeptospirillum

YP 459829, 1 hypothetical protein ELI 14700 [Erythrobacter litoralis

YP 001818081 , 1 conjugative relaxase domain-containing protein [Opitutus terrae

ZP 07745472, 1 conjugative relaxase domain protein iMucilaginibacter paludis

CBA73957.1 conjugal transfer nickase/helicase Tral TArsenophonus nasoniael

YP 002248140.1 hypothetical protein THEYE A0292 I hermodesulfovibrio

ZP 06641691. 1 conserved hypothetical protein TSerratia odorifera DSM 45821

ZP 05056614.1 TrwC relaxase family rVerrucomicrobiae bacterium DG12351

ZP 03723740.1 conjugative relaxase domain protein [Opitutaceae bacterium

YP 004210579.1 conjugative relaxase domain protein rAcidobacterium sp.

YP 001522671 .1 hypothetical protein AMI H0004 rAcaryochloris marina

YP 001573657.1 conjugative relaxase domain-containing protein rBurkholderia

EDZ39038.1 Conjugal protein, TraA rLeptospirillum sp. Group II '5-wav CG'l

YP 001632380.1 conjugal transfer protein TBordetella petrii DSM 128041

ZP 06242489.1 conjugative relaxase domain protein [Victivallis vadensis ATCC

EDZ37956. 1 Conjugal protein, TraA rLeptospirillum sp. Group II '5-way CG'l

YP 004488214.1 conjugative relaxase domain-containing protein TDelftia sp. Csl-

ZP 00208504.1 COG0507: ATP-dependent exoDNAse (exonuclease V), alpha

ACJ47794.1 Tral [Klebsiella pneumoniael

ZP 02730551. 1 TrwC protein TGemmata obscuriglobus UQM 22461

ZP 06244759. 1 TrwC relaxase TVictivallis vadensis ATCC BAA-5481

YP 003022160.1 relaxase TGeobacter sp. M211 >gb|ACT 18402.11 conjugative

YP 001521304.1 hypothetical protein AMI B0272 rAcaryochloris marina

ZP 01091846.1 hypothetical protein DSM3645 02833 TBlastopirellula marina

CAZ881 17.1 putative ATP-dependent exoDNAse (exonuclease V), alpha

YP 004718365.1 conjugative relaxase domain-containing protein TSulfobacillus

YP 002553030.1 conjugative relaxase domain-containing protein [Acidovorax

YP 003386820.1 conjugative relaxase domain-containing protein TSpirosoma

YP 001 Π 9893 , 1 exonuclease V subunit alpha rBurkholderia vietnamiensis G41

YP 315444. 1 putative ATP-dependent exoDNAse (exonuclease V) subunit

YP 003071370.1 hypothetical protein p2METDI0024 [Methyl obacterium

YP 003125939.1 conjugative relaxase [Chitinophaga pinensis DSM 25881

ZP 08495729. 1 TrwC relaxase TMicrocoleus vaginatus FGP-21 >gb|EGK83455.1

EFZ53417.1 tral domain protein [Shigella sonnei 53G1

EGR70910.1 conjugal transfer nickase/helicase Tral [Escherichia coli O104:H4

YP 002912178, 1 ATP-dependent exoDNAse (exonuclease V) subunit alpha

ZP 01089566.1 hypothetical protein DSM3645 27912 TBlastopirellula marina

YP 002753784.1 DNA helicase domain protein rAcidobacterium capsulatum ATCC

EFZ60916.1 protein tral domain protein [Escherichia coli LT-681

ZP 08262009. 1 protein tral [Asticcacaulis biprosthecum C191 >gb|EGF93811.11

AEI11045.1 TrwC relaxase r[Cellvibriol gilvus ATCC 131271

ZP 05040209.1 hypothetical protein S7335 1177 [Synechococcus sp. PCC 73351

YP 001840830, 1 ATP-dependent exoDNAse (exonuclease V) [Mycobacterium

YP 001700713.1 TraA ATP-dependent exoDNAse/relaxase [Mycobacterium

CAC86586.1 conjugal transfer protein [Agrobacterium tumefaciensl

NP 355808.2 conjugation protein [Agrobacterium tumefaciens str. C581

YP 001120496.1 hypothetical protein Beep 1808 2669 [Burkholderia vietnamiensis

EGW76304.1 protein tral domain protein [Escherichia coli STEC B2F11

YP 002979543.1 Ti-type conjugative transfer relaxase TraA [Rhizobium

EHB4404L 1 TrwC relaxase [Mycobacterium rhodesiae JS601

YP 002984810, 1 Ti-type conjugative transfer relaxase TraA [Rhizobium 472 ZP 06848350.1 ATP-dependent exoDNAse (exonuclease V) rMvcobacterium

473 YP 001972793.1 putative conjugal transfer protein TraA rStenotrophomonas

474 ZP 06760230. 1 putative conjugative relaxase domain protein TVeillonella sp.

475 YP 001840914.1 TrwC protein TAcinetobacter baumannii ACICUl

476 YP 003311407.1 TrwC relaxase TVeillonella parvula DSM 20081

477 ZP 08208016.1 TrwC protein TNovosphinsobium nitrogenifigens DSM 193701

478 P 003342708, 1 hypothetical protein SMAC 10304 TSordaria macrospora k-helll

479 YP 004074482, 1 TrwC relaxase rMvcobacterium sp. Spvrll >eb I ADU02001. i l

480 YP 93551 1 , 1 exonuclease V subunit alpha rMvcobacterium sp. KMS1

481 YP 004100308.1 TrwC relaxase ITntrasporangium calvum DSM 430431

482 YP 003326911.1 TrwC relaxase TXvlanimonas cellulosilvtica DSM 158941

483 YP 001136860.1 exonuclease V subunit alpha rMvcobacterium gilvum PYR-

484 YP 001776789.1 coniugative relaxase domain-containing protein

485 NP 862296.1 transfer protein homolog TraA rCorynebacterium glutamicuml

486 YP 001851874.1 ATP-dependent exoDNAse (exonuclease V) rMvcobacterium

487 AAS20144.1 TraA-like protein TArthrobacter aurescensl

488 YP 949993.1 putative TraA-like protein TArthrobacter aurescens TCll

489 YP 004271377.1 TrwC relaxase TPlanctomvces brasiliensis DSM 53051

490 YP 001243088.1 putative ATP-dependent exoDNAse TBradvrhizobium sp.

491 YP 771309, 1 putative conjugal transfer protein TraA TRhizobium

492 ZP 02730298.1 TrwC protein TGemmata obscuriglobus UOM 22461

493 EG061143.1 conjugative relaxase domain protein TAcidithiobacillus sp. GGI-

494 YP 002978744.1 Ti-tvpe conjugative transfer relaxase TraA TRhizobium

495 YP 002973152.1 Ti-tvpe conjugative transfer relaxase TraA TRhizobium

496 ZP 06846967. 1 ATP-dependent exoDNAse (exonuclease V) rMvcobacterium

497 YP 003377696.1 TraA rCorvnebacterium glutamicuml >dbj IB AI66031.11 TraA.

498 ZP 06846356.1 Ti-type conjugative transfer relaxase TraA TBurkholderia sp.

499 YP 001136826.1 exonuclease V subunit alpha rMvcobacterium gilvum PYR-

500 YP 949954.1 putative TraA-like conjugal transfer protein TArthrobacter

The Tral helicase or Tral subgroup helicase is more preferably one of the helicases shown in Table 7 below or a variant thereof. The Tral helicase or Tral subgroup helicase more preferably comprises the sequence of one of the helicases shown in Table 7, i.e. one of SEQ ID NOs: 61, 65, 69, 73, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168, or a variant thereof .

Table 7 - More preferred Tral helicase and Tral subgroup helicases

SEQ ID NOs: 78 and 106 comprise a MobQ motif III, whereas the other sequences in Table 7 comprise a MobF motif III. The Tral helicase preferably comprises the sequence shown in SEQ ID NO: 61 or a variant thereof.

A variant of a RecD helicase is an enzyme that has an amino acid sequence which varies from that of the wild-type helicase and which retains polynucleotide binding activity. In particular, a variant of any one of SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 37, 39, 41, 42, 44, 61, 65, 69, 73, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168 is an enzyme that has an amino acid sequence which varies from that of any one of SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 37, 39, 41, 42, 44, 61, 65, 69, 73, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168 and which retains polynucleotide binding activity. A variant of SEQ ID NO: 18 or 61 is an enzyme that has an amino acid sequence which varies from that of SEQ ID NO: 18 or 61 and which retains polynucleotide binding activity. The variant retains helicase activity. Methods for measuring helicase activity are known in the art. Helicase activity can also be measured as described in the Examples. The variant must work in at least one of the two modes discussed below. Preferably, the variant works in both modes. The variant may include modifications that facilitate handling of the polynucleotide encoding the helicase and/or facilitate its activity at high salt

concentrations and/or room temperature. Variants typically differ from the wild-type helicase in regions outside of the motifs discussed above. However, variants may include modifications within these motif(s).

Over the entire length of the amino acid sequence of any one of SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 37, 39, 41, 42, 44, 61, 65, 69, 73, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168, such as SEQ ID NO: 18 or 61, a variant will preferably be at least 10% homologous to that sequence based on amino acid identity. More preferably, the variant polypeptide may be at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of any one of SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 37, 39, 41, 42, 44, 61, 65, 69, 73, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168, such as SEQ ID NO: 18 or 61, over the entire sequence. There may be at least 70%>, for example at least 80%, at least 85%, at least 90% or at least 95%, amino acid identity over a stretch of 150 or more, for example 200, 300, 400, 500, 600, 700, 800, 900 or 1000 or more, contiguous amino acids ("hard homology"). Homology is determined as described above. The variant may differ from the wild-type sequence in any of the ways discussed above with reference to SEQ ID NOs: 2 and 4.

In particular, variants may include fragments of SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 37, 39, 41, 42, 44, 61, 65, 69, 73, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168. Such fragments retain polynucleotide binding activity. Fragments may be at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 650, at least about 700, at least about 800, at least about 900 or at least about 1000 amino acids in length. The length of the fragment will depend on the length of the wild-type sequence. As discussed in more detail below, fragments preferably comprise the RecD-like motif I and/or the RecD-like motif V of the relevant wild-type sequence.

As discussed above, Tral helicases and Tral subgroup helicases comprise a relaxase domain. The relaxase domain comprises the MobF motif III or the the MobQ motif III and is typically found at the amino (N) terminus of the Tral helicase or Tral subgroup helicase.

Preferred fragments of Tral helicases and Tral subgroup helicases, such as preferred fragments of SEQ ID NOs: 61, 65, 69, 73, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168, lack the N terminal domain of the wild-type sequence. The N-terminal domain typically corresponds to the about the N terminal third of the protein. In SEQ ID NO: 61 (which is 1756 amino acids in length), the N-terminal domain is typically from about 500 to about 700 amino acids in length, such as from about 550 to about 600 amino acids in length. In SEQ ID NOs: 65, 69 and 73 (which are 970, 943 and 960 amino acids in length respectively), the N-terminal domain is typically from about 300 to about 350 amino acids in length, such as from about 320 to about 340 amino acids in length.

Amino acid substitutions may be made to the amino acid sequence of SEQ ID NO: 18,

21, 24, 25, 28, 30, 32, 35, 37, 39, 41, 42, 44, 61, 65, 69, 73, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168, for example up to 1, 2, 3, 4, 5, 10, 20 or 30 substitutions. The substitutions are preferably conservative substitutions as discussed above. One or more substitutions may be made at amino acid positions K555, R554, T644, R647, P666, M667, H646, N604, N596, Y598, V470, G391, H409, T407, R410 and Y414 of SEQ ID NO: 41. In SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 37, 39, 42, 44, 61, 65, 69, 73, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168, substitutions may be made at one or more amino acid positions which correspond to amino acid positions K555, R554, T644, R647, P666, M667, H646, N604, N596, Y598, V470, G391, H409, T407, R410 and Y414 of SEQ ID NO: 41. It is straightforward to determine corresponding amino acid positions in different protein sequences. For instance, the proteins may be aligned based on their homology. Homology may be determined as discussed above.

A variant, such as a fragment, of any one of SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 37, 39, 41, 42, 44, 61, 65, 69, 73, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168 preferably comprises the RecD-like motif I (or RecD motif I) and/or RecD-like motif V (or RecD motif V) of the relevant wild-type sequence. A variant, such as a fragment, of any one of SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 37, 39, 41, 42, 44, 61, 65, 69, 73, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168 preferably comprises the RecD-like motif I (or RecD motif I) and the RecD-like motif V (or RecD motif V) of the relevant wild-type sequence. For instance, a variant of SEQ ID NO: 18 preferably comprises the RecD motif I GGPGTGKT (SEQ ID NO: 19) and the RecD motif V WAVTIHKSQG (SEQ ID NO: 20). The RecD-like motifs I and V (or RecD motifs I and V) of each of SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 37, 39, 41, 42, 44, 61, 65, 69, 73, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168 are shown in Tables 5 and 7. However, a variant of any one SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 37, 39, 41, 42, 44, 61, 65, 69, 73, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168 may comprise the RecD-like motif I (or RecD motif I) and/or RecD-like motif V (or RecD motif V) from a different wild-type sequence. For instance, a variant of SEQ ID NO: 28 or SEQ ID NO: 35 may comprise the RecD motif I and RecD-like motif V of SEQ ID NO: 21 (GGPGTGKS and YALTVHRAQG respectively; SEQ ID NOs: 22 and 23). A variant of any one SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 37, 39, 41, 42, 44, 61, 65, 69, 73, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168 may comprise any one of the preferred motifs shown in Tables 5 and 7. Variants of any one of SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 37, 39, 41, 42, 44, 61, 65, 69, 73, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168 may also include modifications within the RecD-like motifs I and V of the relevant wild-type sequence. Suitable modifications are discussed above when defining the two motifs. The discussion in the paragraph equally applies to the MobF motif III in SEQ ID NOs: 61, 65, 69, 73, 74, 82, 86, 90, 94, 98, 102, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168 and MobQ motif III in SEQ ID NOs: 78 and 106. In particular, a variant, such as a fragment, of any one of SEQ ID NOs: 61, 65, 69, 73, 74, 82, 86, 90, 94, 98, 102, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168 preferably comprises the MobF motif III of the relevant wild-type sequence. A variant, such as a fragment, of SEQ ID NO: 78 or 106 preferably comprises the MobQ motif III of the relevant wild-type sequence. A variant, such as a fragment, of any one of SEQ ID NOs: 61, 65, 69, 73, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168 preferably comprises the RecD-like motif I (or RecD motif I), RecD-like motif V (or RecD motif V) and MobF or MobQ motif III of the relevant wild-type sequence.

The helicase may be covalently attached to the pore. The helicase is preferably not covalently attached to the pore. The application of a voltage to the pore and helicase typically results in the formation of a sensor that is capable of sequencing target polynucleotides. This is discussed in more detail below.

Any of the proteins described herein, i .e. the transmembrane protein pores or RecD helicases, may be modified to assist their identification or purification, for example by the addition of histidine residues (a his tag), aspartic acid residues (an asp tag), a streptavidin tag, a flag tag, a SUMO tag, a GST tag or a MBP tag, or by the addition of a signal sequence to promote their secretion from a cell where the polypeptide does not naturally contain such a sequence. An alternative to introducing a genetic tag is to chemically react a tag onto a native or engineered position on the pore or helicase. An example of this would be to react a gel-shift reagent to a cysteine engineered on the outside of the pore. This has been demonstrated as a method for separating hemolysin hetero-oligomers (Chem Biol. 1997 Jul;4(7):497-505).

The pore and/or helicase may be labelled with a revealing label. The revealing label may be any suitable label which allows the pore to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, e.g. ¹²⁵1, ³⁵S, enzymes, antibodies, antigens, polynucleotides and ligands such as biotin.

Proteins may be made synthetically or by recombinant means. For example, the pore and/or helicase may be synthesized by in vitro translation and transcription (IVTT). The amino acid sequence of the pore and/or helicase may be modified to include non-naturally occurring amino acids or to increase the stability of the protein. When a protein is produced by synthetic means, such amino acids may be introduced during production. The pore and/or helicase may also be altered following either synthetic or recombinant production.

The pore and/or helicase may also be produced using D-amino acids. For instance, the pore or helicase may comprise a mixture of L-amino acids and D-amino acids. This is conventional in the art for producing such proteins or peptides. The pore and/or helicase may also contain other non-specific modifications as long as they do not interfere with pore formation or helicase function. A number of non-specific side chain modifications are known in the art and may be made to the side chains of the protein(s). Such modifications include, for example, reductive alkylation of amino acids by reaction with an aldehyde followed by reduction with NaBH₄, amidination with methylacetimidate or acylation with acetic anhydride.

The pore and helicase can be produced using standard methods known in the art.

Polynucleotide sequences encoding a pore or helicase may be derived and replicated using standard methods in the art. Polynucleotide sequences encoding a pore or helicase may be expressed in a bacterial host cell using standard techniques in the art. The pore and/or helicase may be produced in a cell by in situ expression of the polypeptide from a recombinant expression vector. The expression vector optionally carries an inducible promoter to control the expression of the polypeptide. These methods are described in described in Sambrook, J. and Russell, D. (2001). Molecular Cloning: A Laboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

The pore and/or helicase may be produced in large scale following purification by any protein liquid chromatography system from protein producing organisms or after recombinant expression. Typical protein liquid chromatography systems include FPLC, AKTA systems, the Bio-Cad system, the Bio-Rad BioLogic system and the Gilson HPLC system.

The method of the invention involves measuring one or more characteristics of the target polynucleotide. The method may involve measuring two, three, four or five or more characteristics of the target polynucleotide. The one or more characteristics are preferably selected from (i) the length of the target polynucleotide, (ii) the identity of the target polynucleotide, (iii) the sequence of the target polynucleotide, (iv) the secondary structure of the target polynucleotide and (v) whether or not the target polynucleotide is modified. Any combination of (i) to (v) may be measured in accordance with the invention.

For (i), the length of the polynucleotide may be measured using the number of interactions between the target polynucleotide and the pore.

For (ii), the identity of the polynucleotide may be measured in a number of ways. The identity of the polynucleotide may be measured in conjunction with measurement of the sequence of the target polynucleotide or without measurement of the sequence of the target polynucleotide. The former is straightforward; the polynucleotide is sequenced and thereby identified. The latter may be done in several ways. For instance, the presence of a particular motif in the polynucleotide may be measured (without measuring the remaining sequence of the polynucleotide). Alternatively, the measurement of a particular electrical and/or optical signal in the method may identify the target polynucleotide as coming from a particular source.

For (iii), the sequence of the polynucleotide can be determined as described previously. Suitable sequencing methods, particularly those using electrical measurements, are described in Stoddart D et al., Proc Natl Acad Sci, 12; 106(19):7702-7, Lieberman KR et al, J Am Chem Soc. 2010; 132(50): 17961-72, and International Application WO 2000/28312.

For (iv), the secondary structure may be measured in a variety of ways. For instance, if the method involves an electrical measurement, the secondary structure may be measured using a change in dwell time or a change in current flowing through the pore. This allows regions of single-stranded and double-stranded polynucleotide to be distinguished.

For (v), the presence or absence of any modification may be measured. The method preferably comprises determining whether or not the target polynucleotide is modified by methylation, by oxidation, by damage, with one or more proteins or with one or more labels, tags or spacers. Specific modifications will result in specific interactions with the pore which can be measured using the methods described below. For instance, methylcyotsine may be

distinguished from cytosine on the basis of the current flowing through the pore during its interation with each nucleotide.

A variety of different types of measurements may be made. This includes without limitation: electrical measurements and optical measurements. Possible electrical measurements include: current measurements, impedance measurements, tunnelling measurements (Ivanov AP et al., Nano Lett. 2011 Jan 12; 1 l(l):279-85), and FET measurements (International

Application WO 2005/124888). Optical measurements may be combined 10 with electrical measurements (Soni GV et al., Rev Sci Instrum. 2010 Jan;81(l):014301). The measurement may be a transmembrane current measurement such as measurement of ionic current flowing through the pore.

Electrical measurements may be made using standard single channel recording equipment as describe in Stoddart D et al., Proc Natl Acad Sci, 12; 106(19):7702-7, Lieberman KR et al, J Am Chem Soc. 2010; 132(50): 17961-72, and International Application

WO-2000/28312. Alternatively, electrical measurements may be made using a multi-channel system, for example as described in International Application WO-2009/077734 and

International Application WO-201 1/067559.

In a preferred embodiment, the method comprises:

(a) contacting the target polynucleotide with a transmembrane pore and a RecD helicase such that the target polynucleotide moves through the pore and the RecD helicase controls the movement of the target polynucleotide through the pore; and (b) measuring the current passing through the pore as the polynucleotide moves with respect to the pore wherein the current is indicative of one or more characteristics of the target polynucleotide and thereby characterising the target polynucleotide.

The methods may be carried out using any apparatus that is suitable for investigating a membrane/pore system in which a pore is inserted into a membrane. The method may be carried out using any apparatus that is suitable for transmembrane pore sensing. For example, the apparatus comprises a chamber comprising an aqueous solution and a barrier that separates the chamber into two sections. The barrier has an aperture in which the membrane containing the pore is formed.

The methods may be carried out using the apparatus described in International

Application No. PCT/GB08/000562 (WO 2008/102120).

The methods may involve measuring the current passing through the pore as the polynucleotide moves with respect to the pore. Therefore the apparatus may also comprise an electrical circuit capable of applying a potential and measuring an electrical signal across the membrane and pore. The methods may be carried out using a patch clamp or a voltage clamp. The methods preferably involve the use of a voltage clamp.

The methods of the invention may involve the measuring of a current passing through the pore as the polynucleotide moves with respect to the pore. Suitable conditions for measuring ionic currents through transmembrane protein pores are known in the art and disclosed in the Example. The method is typically carried out with a voltage applied across the membrane and pore. The voltage used is typically from +2 V to -2 V, typically -400 mV to +400mV. The voltage used is preferably in a range having a lower limit selected from -400 mV, -300 mV, -200 mV, -150 mV, -100 mV, -50 mV, -20mV and 0 mV and an upper limit independently selected from +10 mV, + 20 mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mV and +400 mV. The voltage used is more preferably in the range 100 mV to 240mV and most preferably in the range of 120 mV to 220 mV. It is possible to increase discrimination between different nucleotides by a pore by using an increased applied potential.

The methods are typically carried out in the presence of any charge carriers, such as metal salts, for example alkali metal salt, halide salts, for example chloride salts, such as alkali metal chloride salt. Charge carriers may include ionic liquids or organic salts, for example tetramethyl ammonium chloride, trimethylphenyl ammonium chloride, phenyltrimethyl ammonium chloride, or l-ethyl-3 -methyl imidazolium chloride. In the exemplary apparatus discussed above, the salt is present in the aqueous solution in the chamber Potassium chloride (KC1), sodium chloride (NaCl) or caesium chloride (CsCl) is typically used. KC1 is preferred. The salt concentration may be at saturation. The salt concentration may be 3M or lower and is typically from 0.1 to 2.5 M, from 0.3 to 1.9 M, from 0.5 to 1.8 M, from 0.7 to 1.7 M, from 0.9 to 1.6 M or from 1 M to 1.4 M. The salt concentration is preferably from 150 mM to 1 M. As discussed above, RecD helicases surprisingly work under high salt concentrations. The method is preferably carried out using a salt concentration of at least 0.3 M, such as at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M or at least 3.0 M. High salt concentrations provide a high signal to noise ratio and allow for currents indicative of the presence of a nucleotide to be identified against the background of normal current fluctuations.

The methods are typically carried out in the presence of a buffer. In the exemplary apparatus discussed above, the buffer is present in the aqueous solution in the chamber. Any buffer may be used in the method of the invention. Typically, the buffer is HEPES. Another suitable buffer is Tris-HCl buffer. The methods are typically carried out at a pH of from 4.0 to 12.0, from 4.5 to 10.0, from 5.0 to 9.0, from 5.5 to 8.8, from 6.0 to 8.7 or from 7.0 to 8.8 or 7.5 to 8.5. The pH used is preferably about 7.5.

The methods may be carried out at from 0 °C to 100 °C, from 15 °C to 95 °C, from 16 °C to 90 °C, from 17 °C to 85 °C, from 18 °C to 80 °C, 19 °C to 70 °C, or from 20 °C to 60 °C. The methods are typically carried out at room temperature. The methods are optionally carried out at a temperature that supports enzyme function, such as about 37 °C.

The method is typically carried out in the presence of free nucleotides or free nucleotide analogues and an enzyme cofactor that facilitate the action of the helicase. The free nucleotides may be one or more of any of the individual nucleotides discussed above. The free nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine

diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP). The free nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP. The free nucleotides are preferably adenosine triphosphate (ATP). The enzyme cofactor is a factor that allows the helicase to function. The enzyme cofactor is preferably a divalent metal cation. The divalent metal cation is preferably Mg , Mn , Ca or Co . The enzyme cofactor is most preferably Mg²⁺.

The target polynucleotide may be contacted with the RecD helicase and the pore in any order. In is preferred that, when the target polynucleotide is contacted with the RecD helicase and the pore, the target polynucleotide firstly forms a complex with the helicase. When the voltage is applied across the pore, the target polynucleotide/helicase complex then forms a complex with the pore and controls the movement of the polynucleotide through the pore.

As discussed above, RecD helicases may work in two modes with respect to the pore.

First, the method is preferably carried out using the RecD helicase such that it moves the target sequence through the pore with the field resulting from the applied voltage. In this mode the 5' end of the DNA is first captured in the pore, and the enzyme moves the DNA into the pore such that the target sequence is passed through the pore with the field until it finally translocates through to the trans side of the bilayer. Alternatively, the method is preferably carried out such that the enzyme moves the target sequence through the pore against the field resulting from the applied voltage. In this mode the 3' end of the DNA is first captured in the pore, and the enzyme moves the DNA through the pore such that the target sequence is pulled out of the pore against the applied field until finally ejected back to the cis side of the bilayer.

The method of the invention most preferably involves a pore derived from MspA and a helicase comprising the sequence shown in SEQ ID NO: 61 or a variant thereof. Any of the embodiments discussed above with reference to MspA and SEQ ID NO: 61 may be used in combination. Other methods

The invention also provides a method of forming a sensor for characterising a target polynucleotide. The method comprises forming a complex between a pore and a RecD helicase. The complex may be formed by contacting the pore and the helicase in the presence of the target polynucleotide and then applying a potential across the pore. The applied potential may be a chemical potential or a voltage potential as described above. Alternatively, the complex may be formed by covalently attaching the pore to the helicase. Methods for covalent attachment are known in the art and disclosed, for example, in International Application Nos.

PCT/GB09/001679 (published as WO 2010/004265) and PCT/GB 10/000133 (published as WO 2010/086603). The complex is a sensor for characterising the target polynucleotide. The method preferably comprises forming a complex between a pore derived from Msp and a RecD helicase. Any of the embodiments discussed above with reference to the method of the invention equally apply to this method.

Kits

The present invention also provides kits for characterising a target polynucleotide. The kits comprise (a) a pore and (b) a RecD helicase. Any of the embodiments discussed above with reference to the method of the invention equally apply to the kits.

The kit may further comprise the components of a membrane, such as the phospholipids needed to form an amphiphilic layer, such as a lipid bilayer.

The kits of the invention may additionally comprise one or more other reagents or instruments which enable any of the embodiments mentioned above to be carried out. Such reagents or instruments include one or more of the following: suitable buffer(s) (aqueous solutions), means to obtain a sample from a subject (such as a vessel or an instrument comprising a needle), means to amplify and/or express polynucleotides, a membrane as defined above or voltage or patch clamp apparatus. Reagents may be present in the kit in a dry state such that a fluid sample resuspends the reagents. The kit may also, optionally, comprise instructions to enable the kit to be used in the method of the invention or details regarding which patients the method may be used for. The kit may, optionally, comprise nucleotides. Apparatus

The invention also provides an apparatus for characterising a target polynucleotide. The apparatus comprises a plurality of pores and a plurality of a RecD helicase. The apparatus preferably further comprises instructions for carrying out the method of the invention. The apparatus may be any conventional apparatus for polynucleotide analysis, such as an array or a chip. Any of the embodiments discussed above with reference to the methods of the invention are equally applicable to the apparatus of the invention.

The apparatus is preferably set up to carry out the method of the invention.

The apparatus preferably comprises:

a sensor device that is capable of supporting the membrane and plurality of pores and being operable to perform polynucleotide characterising using the pores and helicases;

at least one reservoir for holding material for performing the characterising;

a fluidics system configured to controllably supply material from the at least one reservoir to the sensor device; and

a plurality of containers for receiving respective samples, the fluidics system being configured to supply the samples selectively from the containers to the sensor device. The apparatus may be any of those described in International Application No. No.

PCT/GB08/004127 (published as WO 2009/077734), PCT/GB 10/000789 (published as WO 2010/122293), International Application No. PCT/GB 10/002206 (not yet published) or

International Application No. PCT/US99/25679 (published as WO 00/28312).

Characterisation without a pore

In some embodiments, the target polynucleotide is characterised, such as partially or completely sequenced, using a RecD helicase, but without using a pore. In particular, the invention also provides a method of characterising a target polynucleotide which comprises contacting the target polynucleotide with a RecD helicase such that the RecD helicase controls the movement of the target polynucleotide. In this method, the target polynucleoide is preferably not contacted with a pore, such as a transmembrane pore. The method involves taking one or more measurements as the RecD helicase controls the movement of the polynucleotide and thereby characterising the target polynucleotide. The measurements are indicative of one or more characteristics of the target polynucleotide. Any such measurements may be taken in accordance with the invention. They include without limitation: electrical measurements and optical measurements. These are discussed in detail above. Any of the embodiments discussed above with reference to the pore-based method of the invention may be used in the method lacking a pore. For instance, any of the RecD helicases discussed above may be used.

The invention also provides an analysis apparatus comprising a RecD helicase. The invention also provides a kit a for characterising a target polynucleotide comprising (a) an analysis apparatus for characterising target polynucleotides and (b) a RecD helicase. These apparatus and kits preferably do not comprise a pore, such as a transmembrane pore. Suitable apparatus are discussed above.

The following Examples illustrate the invention.

Example 1

This example illustrates the use of a Tral helicase (Tral Eco; SEQ ID NO: 61) to control the movement of intact DNA strands through a nanopore. The general method and substrate employed throughout this example is shown in Fig. 1 and described in the figure caption

Materials and Methods

Primers were designed to amplify a -400 bp fragment of PhiX174. Each of the 5 '-ends of these primers included a 50 nucleotide non-complimentary region, either a homopolymeric stretch or repeating units of 10 nucleotide homopolymeric sections. These serve as identifiers for controlled translocation of the strand through a nanopore, as well as determining the directionality of translocation. In addition, the 5 '-end of the forward primer was "capped" to include four 2'-0-Methyl-Uracil (mU) nucleotides and the 5 '-end of the reverse primer was chemically phosphorylated. These primer modifications then allow for the controlled digestion of predominantly only the antisense strand, using lambda exonuclease. The mU capping protects the sense strand from nuclease digestion whilst the P04 at the 5' of the antisense strand promotes it. Therefore after incubation with lambda exonuclease only the sense strand of the duplex remains intact, now as single stranded DNA (ssDNA). The generated ssDNA was then PAGE purified as previously described.

The DNA substrate design used in all the experiments described here is shown in Fig. IB. The DNA substrate consists of a 400 base section of ssDNA from PhiX, with a 50T 5 '-leader to aid capture by the nanopore (SEQ ID NO: 172). Annealed to this strand just after the 50T leader is a primer (SEQ ID NO: 173) containing a 3' cholesterol tag to enrich the DNA on the surface of the bilayer, and thus improve capture efficiency. An additional primer (SEQ ID NO: 174) is used towards the 3 ' end of the strand to aid the capture of the strand by the 3 ' end.

Buffered solution: 400 mM NaCl, 10 mM Hepes, pH 8.0, 1 mM ATP, 1 mM MgCl₂, 1 mM DTT

Nanopore: E.coli MS(B2)8 MspA ONLP3476 MS-(L88N/D90N/D91N/D93N/

D118R/D134R/E139K)8

Enzyme: Tral Eco (SEQ ID NO: 61; ONLP3572, ~4. 3 μΜ) 23.3 μΐ -> 100 nM final.

Electrical measurements were acquired from single MspA nanopores inserted in 1,2- diphytanoyl-glycero-3-phosphocholine lipid (Avanti Polar Lipids) bilayers. Bilayers were formed across -100 μιη diameter apertures in 20 μπι thick PTFE films (in custom Delrin chambers) via the Montal-Mueller technique, separating two 1 mL buffered solutions. All experiments were carried out in the stated buffered solution. Single-channel currents were measured on Axopatch 200B amplifiers (Molecular Devices) equipped with 1440A digitizers. Ag/AgCl electrodes were connected to the buffered solutions so that the cis compartment (to which both nanopore and enzyme/DNA are added) is connected to the ground of the Axopatch headstage, and the trans compartment is connected to the active electrode of the headstage. After achieving a single pore in the bilayer, DNA polynucleotide and helicase were added to 50 \iL of buffer and pre-incubated for 5 mins (DNA = 12.0 nM, Enzyme = 2 μΜ). This pre-incubation mix was added to 950 μΐ_^ of buffer in the cis compartment of the electrophysiology chamber to initiate capture of the helicase-DNA complexes in the MspA nanopore (to give final concentrations of DNA = 0.6 nM, Enzyme = 0.1 μΜ). Helicase ATPase activity was initiated as required by the addition of divalent metal (1 mM MgCl₂) and NTP (1 mM ATP) to the cis compartment. Experiments were carried out at a constant potential of +140 mV.

Results and Discussion

The addition of Helicase-DNA substrate to MspA nanopores as shown in Fig. 1 produces characteristic current blocks as shown in Figs. 2 and 3. DNA without helicase bound interacts transiently with the nanopore producing short-lived blocks in current (« 1 second). DNA with helicase bound and active (ie. moving along the DNA strand under ATPase action) produces long characteristic block levels with stepwise changes in current as shown in Figs. 2 and 3.

Different DNA motifs in the nanopore give rise to unique current block levels.

For a given substrate, we observe a characteristic pattern of current transitions that reflects the

DNA sequence (examples in Fig. 3).

In the implementation shown in Fig. 1, the DNA strand is sequenced from a random starting point as the DNA is captured with a helicase at a random position along the strand.

Salt tolerance

Nanopore strand sequencing experiments of this type generally require ionic salts. The ionic salts are necessary to create a conductive solution for applying a voltage offset to capture and translocate DNA, and to measure the resulting sequence dependent current changes as the DNA passes through the nanopore. Since the measurement signal is dependent on the concentration of the ions, it is advantageous to use high concentration ionic salts to increase the magnitude of the acquired signal. For nanopore sequencing salt concentrations in excess of 100 mM KC1 are ideal, and salt concentrations of 400mM KC1 and above are preferred.

However, many enzymes (including some helicases and DNA motor proteins) do not tolerate high salt conditions. Under high salt conditions the enzymes either unfold or lose structural integrity, or fail to function properly. The current literature for known and studied helicases shows that almost all helicases fail to function above salt concentrations of approximately 100 mM KCl/NaCl, and there are no reported helicases that show correct activity in conditions of 400 mM KC1 and above. While potentially halophilic variants of similar enzymes from halotolerant species exist, they are extremely difficult to express and purify in standard expression systems (e g. E. coli). We surprisingly show in this Example that Tral displays salt tolerance up to very high levels of salt. We find that the enzyme retains functionality in salt concentrations of 400 mM KC1 through to 1 M KC1, either in fluorescence experiments or in nanopore experiments. Forward and reverse modes of operation

Most helicases move along single-stranded polynucleotide substrates in uni-directional manner, moving a specific number of bases for each NTPase turned over. Helicase movement can be exploited in different modes to feed DNA through the nanopore in a controlled fashion. Fig. 1 illustrates two basic 'forward' and 'reverse' modes of operation. In the forward mode, the DNA is fed into the pore by the helicase in the same direction as the DNA would move under the force of the applied field. This direction is shown by the trans arrows. For Tral, which is a 5'-3' helicase, this requires capturing the 5' end of the DNA in the nanopore until a helicase contacts the top of the nanopore, and the DNA is then fed into the nanopore under the control of the helicase with the field from the applied potential, ie. moving from cis to trans. The reverse mode requires capturing the 3 ' end of the DNA, after which the helicase proceeds to pull the threaded DNA back out of the nanopore against the field from the applied potential, ie. moving from trans to cis. Fig.1 shows these two modes of operation using Tral Eco.

Example 2

This example illustrates the salt tolerance of RecD helicases using a fluorescence assay for testing enzyme activity.

A custom fluorescent substrate was used to assay the ability of the helicase to displace hybridised dsDNA (Fig. 4A). As shown in 1) of Fig. 4A, the fluorescent substrate strand (50 nM final) has a 5' ssDNA overhang, and a 40 base section of hybridised dsDNA. The major upper strand has a carboxyfluorescein base at the 3 ' end, and the hybrised complement has a black-hole quencher (BHQ-1) base at the 5' end. When hybrised the fluorescence from the fluorescein is quenched by the local BHQ-1, and the substrate is essentially non- fluorescent. 1 μΜ of a capture strand that is complementary to the shorter strand of the fluorescent substrate is included in the assay. As shown in 2), in the presence of ATP (1 mM) and MgCl₂ (10 mM), helicase (100 nM) added to the substrate binds to the 5' tail of the fluorescent substrate, moves along the major strand, and displaces the complementary strand as shown. As shown in 3), once the

complementary strand with BHQ-1 is fully displaced the fluorescein on the major strand fluoresces. As shown in 4), an excess of capture strand preferentially anneals to the

complementary DNA to prevent re-annealing of initial substrate and loss of fluorescence. Substrate DNA: SEQ ID NO: 175 with a carboxyfluorescein near the 3' end and SEQ ID NO: 176 with a Black Hole Quencher- 1 at the 5' end

Capture DNA: SEQ ID NO: 177 The graph in Fig. 4B shows the initial rate of activity of two RecD helicases (RecD Nth and Dth, SEQ IDs 28 and 35) in buffer solutions (100 mM Hepes pH 8.0, 1 mM ATP, 10 mM MgC , 50 nM fluorescent substrate DNA, 1 μΜ capture DNA) containing different

concentrations of KC1 from 100 mM to 1 M. The helicase works at 1 M. Example 3

In this Example, a different Tral helicase was used, namely TrwC Cba (SEQ ID NO: 65). All experiments were carried out as previously described in Example 1 under the same experimental conditions (pore = MspA B2, DNA = 400mer SEQ ID NO: 172, 173 and 174, buffer = 400mM KC1, lOmM Hepes pH 8.0, lmM DTT, ImM ATP, ImM MgCl₂). Fig. 5 shows two typical examples of helicase controlled DNA events using this enzyme.

Example 4

In this Example a number of different TrwC helicases (TrwC (Atr) (SEQ ID NO: 144), TrwC (Sal) (SEQ ID NO: 140), TrwC (Ccr) (SEQ ID NO: 136) and TrwC (Eco) (SEQ ID NO: 74)) were investigated for their ability to control the movement of DNA (SEQ ID NOs: 178, 179 (with /iSpl8//iSpl8//iSp l 8//iSpl 8//iSpl 8//iSpl 8/TT/3CholTEG/ at the 3 ' end) and 180) through an MspA nanopore (MS-

(G75S/G77S/L88N/D90N/D91N/D93N/D1 18R/Q126R/D134R/E139K)8, i.e. 8 x SEQ ID NO: 2 with G75S/G77S/L88N/Q126R.

Materials and Methods

Buffered solution: 625mM KCl, 75mM K Ferrocyanide, 25mM K Ferricyanide, lOOmM Hepes at pH 8.0 for TrwC (Atr),TrwC (Eco) and TrwC (CcR), and at pH 9.0 for TrwC (Sal).

Enzyme: TrwC (Atr) (100 nM) or TrwC (Sal) (100 nM) or TrwC (Ccr) (100 nM) or TrwC (Eco) (100 nM) all at a final concentration of 100 nM

Electrical measurements were acquired from single MspA nanopores inserted in 1,2- diphytanoyl-glycero-3-phosphocholine lipid (Avanti Polar Lipids) bilayers as described in Example 1, except platinum electrodes were used instead of Ag/AgCl. After achieving a single pore in the bilayer, MgCl₂ (10 mM) and dTTP (5 mM, for TrwC (Atr), TrwC (Ccr) and TrwC (Eco)) or ATP (1 mM for TrwC (Sal)) were added to the cis chamber and a control experiment was run for 5 mins at an applied potential of +120 mV. DNA polynucleotide (SEQ ID NO: 178 hybridized to 179 and 180, 0.1 nM) was added to the cis chamber and another control experiment was run for 5 mins at an applied potential of +120 mV. Finally, the appropriate helicase (TrwC (Atr), TrwC (Sal), TrwC (Ccr) or TrwC (Eco) all added at a final concentration of 100 nM) was added to the cis compartment of the electrophysiology chamber to initiate capture of the helicase-DNA complexes in the MspA nanopore. Experiments were carried out at a constant potential of +120 mV. Results and Discussion

Helicase controlled DNA movement was observed for each of the helicases investigated.

Example traces are shown in Figures 6-9 respectively. Example 5

In this example, a number of different TrwC helicases (TrwC (Oma) (SEQ ID NO: 106), TrwC (Afe) (SEQ ID NO: 86), and TrwC (Mph) (SEQ ID NO: 94)) were investigated for their ability to control the movement of DNA (SEQ ID NOs: 172 to 174 for TrwC (Oma), and SEQ ID NO: 181 hybridized to SEQ ID NO: 182 (with a cholesterol tag at the 3 ' end) for TrwC (Afe) and TrwC (Mph)) through an MspA nanopore (MS-

(G75S/G77S/L88N/D90N/D91N/T 93N/D1 18R/Q126R/D134R/E139K)8 i.e. 8 x SEQ ID NO: 2 with G75S/G77S/L88N/Q126R.

Buffered solution: 625mM KCl, 75mM K Ferrocyanide, 25mM K Ferricyanide, lOOmM Hepes. pH8.0

Enzyme: TrwC (Oma), TrwC (Afe), and TrwC (Mph) all at a final concentration of 100 nM

Electrical measurements were acquired from single MspA nanopores inserted in 1,2- diphytanoyl-glycero-3-phosphocholine lipid (Avanti Polar Lipids) bilayers as described in Example 1, except platinum electrodes were used instead of Ag/AgCl. After achieving a single pore in the bilayer, MgCl₂ (10 mM) were added to the cis chamber and a control experiment was run for 5 mins at an applied potential of 120 mV. 0.15nM final of DNA polynucleotide (SEQ ID NOs: 172 to 174 (as in Example 1) for TrwC (Oma), or SEQ ID NO: 181 hybridized to 182 for TrwC (Afe) and TrwC (Mph)) and ΙΟΟηΜ final of the appropriate helicase (TrwC (Oma), TrwC (Afe), and TrwC (Mph) were added to the cis chamber and another control experiment was run for 10 mins at an applied potential of +120 mV. Finally, helicase ATPase activity was initiated by the addition of ATP (1 mM) to the cis compartment of the electrophysiology chamber.

Experiments were carried out at a constant potential of +120 mV. Results and Discussion

Helicase controlled DNA movement was observed for each of the helicases investigated. Example traces are shown in Figures 10-12 respectively.

Claims

1. A method of characterising a target polynucleotide, comprising:

(a) contacting the target polynucleotide with a transmembrane pore and a RecD helicase such that the target polynucleotide moves through the pore and the RecD helicase controls the movement of the target polynucleotide through the pore; and

2. A method according to claim 1, wherein the one or more characteristics are selected from (i) the length of the target polynucleotide, (ii) the identity of the target polynucleotide, (iii) the sequence of the target polynucleotide, (iv) the secondary structure of of the target polynucleotide and (v) whether or not the target polynucleotide is modified.

3. A method according to claim 2, wherein the target polynucleotide is modified by methylation, by oxidation, by damage, with one or more proteins or with one or more labels, tags or spacers.

4. A method according to any one of claims 1 to 3, wherein the one or more characteristics of the target polynucleotide are measured by electrical measurement and/or optical measurement.

5. A method according to claim 4, wherein the electrical measurement is a current measurement, an impedance measurement, a tunnelling measurement or a field effect transistor (FET) measurement.

6. A method according to claim 1, wherein the method comprises:

(b) measuring the current passing through the pore as the polynucleotide moves with respect to the pore wherein the current is indicative of one or more characteristics of the target polynucleotide and thereby characterising the target polynucleotide.

7. A method according to any one of the preceding claims, wherein step (b) involves taking one or more measurements as the polynucleotide moves through the pore.

8. A method according to any one of the preceding claims, wherein the method further comprises the step of applying a voltage across the pore to form a complex between the pore and the helicase

9. A method according to any one of the preceding claims, wherein at least a portion of the polynucleotide is double stranded.

10. A method according to any one of the preceding claims, wherein the pore is a transmembrane protein pore or a solid state pore.

11. A method according to claim 10, wherein the transmembrane protein pore is selected from a hemolysin, leukocidin, Mycobacterium smegmatis porin A (MspA), outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, Neisseria autotransporter lipoprotein (NalP) and WZA.

12. A method according to claim 1 1, wherein the transmembrane protein is (a) formed of eight identical subunits as shown in SEQ ID NO: 2 or (b) a variant thereof in which one or more of the seven subunits has at least 50% homology to SEQ ID NO: 2 based on amino acid identity over the entire sequence and retains pore activity.

13. A method according to claim 11, wherein the transmembrane protein is (a) a-hemolysin formed of seven identical subunits as shown in SEQ ID NO: 4 or (b) a variant thereof in which one or more of the seven subunits has at least 50% homology to SEQ ID NO: 4 based on amino acid identity over the entire sequence and retains pore activity.

14. A method according to any one of the preceding claims, wherein the RecD helicase comprises:

- the amino acid motif X1 X2-X3-G-X4-X5-X6-X7 (SEQ ID NO: 8), wherein XI is G, S or A, X2 is any amino acid, X3 is P, A, S or G, X4 is T, A, V, S or C, X5 is G or A, X6 is K or R and X7 is T or S; and/or - the ammo acid motif X 1-X2-X3-X4-X5-(X6)₃-Q-X7 (SEQ ID Os: 9, 10 and 11 ), wherein XI is Y, W or F, X2 is A, T, S. M, C or V, X3 is any amino acid, X4 is T or N, X5 is A, T, G, S, V or I, X6 s any amino acid and X7 is G or S.

1.5. A method according to claim 14, wherein the RecD helicase comprises the following motifs:

(a) GGPGTGKT (SEQ ID NO: 19) and/or WAVTIHKSQG (SEQ ID NO: 20);

(b) GGPGTGKS (SEQ ID NO: 22) and/or YALTVHRAQG (SEQ ID NO: 23);

(c) GGPGVGKT (SEQ ID NO: 26) and/or YAISVHKSQG (SEQ ID NO: 27);

(d) GGPGTGKT (SEQ ID NO: 19) and/or YCISVHKSQG SEQ ID NO: (29);

(e) GGPGVGKT (SEQ ID NO : 26) and/or YAATIHKSQG (SEQ ID NO : 31 );

(f) GGPGCGKS (SEQ ID NO: 33) and/or YAMTHTRSQG (SEQ ID NO: 34);

(g) GGPGTGKS (SEQ ID NO: 22) and/or YAVSIHKSQG (SEQ ID NO: 36);

(h) GGPGVGKT (SEQ ID NO: 26) and/or YATSVHKSQG (SEQ ID NO: 38);

(i) GGPGTGKT (SEQ ID NO: 19) and/or YAVSVHKSQG (SEQ ID NO: 40); (j) GGPGVGKT (SEQ ID NO: 26) and/or YATSIHKSQG (SEQ ID NO: 43); or (k) GGPGTGKS (SEQ ID NO: 22) and/or YALTVHRGQG (SEQ ID NO: 45).

16. A method according to any one of the preceding claims, wherein the RecD helicase is one of the helicases shown in Table 4 or 5 or a variant thereof.

17. A method according to claim 16, wherein the RecD helicase comprises:

(a) the sequence shown in any one of SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 37, 39, 41, 42 and 44; or

(b) a variant thereof having at least 25% homology to the relevant sequence based on amino acid identity over the entire sequence and which retains helicase activity.

18. A method according to any one of claims 1 to 14, wherein the RecD helicase is a Tral helicase or Tral subgroup helicase.

19. A method according to claim 18, wherein the Tral helicase or Tral subgroup helicase further comprises:

- the amino acid motif H X1)₂-X2-R-(X3)_5-12-H-X4-H (SEQ ID NOs: 46 to 53), wherein XI and X3 are any amino acid and X2 and X4 are independently selected from any amino acid except D, E, K and ; or - the amino acid motif G-Xl -Χ2-Χ3-Χ4-Χ5-Χ6-Χ7-Β-(Χ8 ..ι₂-Β-Χ9 (SEQ ID NOs: 54 to 60), wherein XI, X2, X3, X5, X6, X7 and X9 are independently selected from any amino acid except D, E, K and R, X4 is D or E and X8 is any amino acid.

20. A method according to claim 14, wherein the Tral hdicase or Tral subgroup helicase comprises the following motifs:

(a) GYAGVGKT (SEQ ID NO: 62), YA3TAHGAQG (SEQ ID NO: 63) and HDTSRDQEPQLHTH (SEQ ID NO: 64);

(b) GIAGAGKS (SEQ ID NO: 66), YALNVHMAQG (SEQ ID NO: 67) and HDTNRNQEPNLHFH (SEQ ID NO: 68);

( C i GAAGAG T (SEQ ID NO: 70), YCITIHRSQG (SEQ ID NO: 71) and

HEDARTVDDIADPQLHTH (SEQ ID NO: 72);

(d) GFAGTGKS (SEQ SD NO: 75), YATTVHSSQG (SEQ ID NO: 76) and

HETSRERDPQLHTH (SEQ ID NO: 77);

(e) GRAGAGKT (SEQ ID NO: 79), V \ T ri H SQG (SEQ ID NO: 80) and

GMVADWVYHDNPGNPHIH (SEQ ID NO: 81);

(f) G AAGTG T (SEQ ID NO: 83), Y \S i^' \ ! i SQG (SEQ ID NO: 84) and HSTSRAQDPHLHSH (SEQ ID NO: 85);

(g) GHAGAGKT (SEQ ID NO: 87), YAGTTHRNQG (SEQ ID NO: 88) and HA S SREQDPQIHSH (SEQ ID NO: 89);

(h) GLAGTGKT (SEQ ID NO: 91 ), YAVTSHSSQG (SEQ ID NO: 92) and

HDTARPVNGYAAPQLHTH (SEQ ID NO: 93);

(i) GPAGAGKT (SEQ ID NO: 95), YAITAHRAQG (SEQ ID NO: 96) and HYDSRAGDPQLHTH (SEQ ID NO: 97);

(j) GWAGVGKT (SEQ ID NO: 99), YAVTADHMQG (SEQ ID NO: 100) and HLCGRLDPQIHNH (SEQ ID NO: 101);

(k) GVAGAGKT (SEQ ID NO: 103 ), YALTIDSAQG (SEQ ID NO: 104) and HMTSGDGSPHLHVH (SEQ ID NO: 105),

(1) GY AGTGKS (SEQ ID NO: 107), YAATIHKAQG (SEQ ID NO: 108) and GMIADLVNVI-IWDIGEDGKAKPHAl-l (SEQ ID NO: 109);

(m) GIAGAGKS (SEQ ID NO: 66), YALNAHMAQG (SEQ ID NO: 67) and HDTNRNQEPNLHFH (SEQ ID NO: i 11);

(n) GVAGAGKS (SEQ ID NO: 115), YALNAHMAQG (SEQ ID NO: 67) and HDTNRNQEPNAHFH (SEQ ID NO: 116); (o) GGAGVG KS (SEQ ID NO: 118), YAI VHIAQG (SEQ ID NO: 1 19) and HDVSRNNDPQLHVH (SEQ ID NO: 120);

(p) Gi AGAG S (SEQ ID NO: 66), YALNMHMAQG (SEQ ID NO: 122) and HDTSRALDPQGHIH (SEQ ID NO: 123);

(q) GV GAGKS (SEQ ID NO: 115), YALNAHMAQG (SEQ ID NO: 67) and HDTSRALDPQGH!H (SEQ ID NO: 123);

(r) GRAGTGKT (SEQ ID NO: 126), FASTAHGAQG (SEQ ID NO: 127) and

BEASRNLDPQLBSB (SEQ ID NO-. 128);

(s) GYAGTGKT (SEQ ID NO: 130), YAMTSHAAQG (SEQ ID NO: 131) and HDIS DKDPQLHTH (SEQ ID NO: 1 2);

(t) GLAGTGKT (SEQ ID NO: 91). YAQTVHASQG (SEQ ID NO: 134) and HNTSRDLDPQTBTH (SEQ ID NO: 135);

(u) GFAGTAKT (SEQ ID NO: 137), YVQTAFAAQG (SEQ ID NO: 138) and HETSRAQDPQLHTB (SEQ ID NO: 1 9);

(v) GY AGT \ Γ (SEQ ID NO: 141), YVDTAF AQG (SEQ ID NO: 142) and HGTSRAQDPQLHTH (SEQ ID NO: 143);

(w) GYAGTAKT (SEQ ID NO: 141), YASTAFAAQG (SEQ ID NO: 145) and HGTSRALDPQLHSH (SEQ ID NO: 146);

(x) GSAGSGKT (SEQ ID NO: 148), YAVTSYSAQG (SEQ ID NO: 149) and HDIAI^VGGYAAPQLHTH (SEQ ID NO: 150);

(y) GEAGTGKT (SEQ ID NO: 153), YAHTS YKEQG (SEQ ID NO: 1 4) and HETNRENEPQLHNH ( SEQ ID NO: 155);

(z) GYAGVAKT (SEQ ID NO: 157), Y TTNYKVQG (SEQ LD NO: 158) and QPSSRANDPALBTB (SEQ ID NO: 1 59);

(aa) GSAGTGKT (SEQ ID NO: 161), YSLTANRAQG (SEQ LD NO: 162) and BSMSRAGDPEMHNH (SEQ ID NO: 163),

(bb) AGAGTGKT (SEQ ID NO: 165), YAGTVYAAQG (SEQ ID NO: 166) and HYTTREGDPNIHTH (SEQ ID NO: 167); or

(cc) APAGAGKT (SEQ ID NO: 169), YAVTVHAAQG (SEQ ID NO: 170) and HETSRAGDPHLHTH (SEQ ID NO: 171).

21. A method according to any one of claims 18 to 20, wherein the Tral helicase or Tral subgroup helicase is one of the helicases shown in Table 6 or 7 or a variant thereof.

22. A method according to claim 21, wherein the Tral helicase or Tral subgroup helicase comprises (a) the sequence shown in any one of SEQ ID NOs: 61, 65, 69, 73, 74, 78, 82, 86, 90, 94, 98, 102, 106, 1 10, 1 12, 1 13, 1 14, 1 17, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168 or (b) a variant thereof having at least 10% homology to the relevant sequence based on amino acid identity over the entire sequence and retains helicase activity.

23. A method according to any one of the preceding claims, wherein the method is carried out using a salt concentration of at least 0.3 M and the salt is optionally KC1.

24. A method according to claim 23, wherein the salt concentration is at least 1.0 M.

25. A method of forming a sensor for characterising a target polynucleotide, comprising forming a complex between a pore and a RecD helicase and thereby forming a sensor for characterising the target polynucleotide.

26. A method according to claim 25, wherein the complex is formed by (a) contacting the pore and the helicase in the presence of the target polynucleotide and (a) applying a potential across the pore.

27. A method according to claim 26, wherein the potential is a voltage potential or a chemical potential.

28. A method according to claim 26, wherein the complex is formed by covalently attaching the pore to the helicase.

29. Use of a RecD helicase to control the movement of a target polynucleotide through a pore.

30. A kit for characterising a target polynucleotide comprising (a) a pore and (b) a RecD helicase.

31. A kit according to claim 29, wherein the kit further comprises a chip comprising an amphiphilic layer.

32. An analysis apparatus for characterising target polynucleotides in a sample, comprising a plurality of pores and a plurality of a RecD helicase.

33. An analysis apparatus according to claim 32, wherein the analysis apparatus comprises: a sensor device that is capable of supporting the plurality of pores and being operable to perform polynucleotide characterisation using the pores and helicases;

at least one reservoir for holding material for performing the characterisation;

a plurality of containers for receiving respective samples, the fluidics system being configured to supply the samples selectively from the containers to the sensor device.

34. A method of characterising a target polynucleotide, comprising:

(b) taking one or more measurements as the RecD helicase controls the movement of the polynucleotide wherein the measurements are indicative of one or more characteristics of the target polynucleotide and thereby characterising the target polynucleotide.

35. A method according to claim 34, wherein:

(a) the one or more characteristics are as defined in claim 2;

(b) the target polynucleotide is as defined in claim 3 or 9;

(c) the one or more characteristics are measured as defined in claim 4 or 5;

(d) the RecD is as defined in any one of claims 14 to 22; or

(e) the method is carried out as defined in any claim 23 or 24.

36. Use of a RecD helicase to control the movement of a target polynucleotide during characterisation of the polynucleotide.

37. Use of a RecD helicase to control the movement of a target polynucleotide during sequencing of part or all of the polynucleotide.

38. An analysis apparatus for characterising target polynucleotides in a sample, characterised in that it comprises a RecD helicase.

39. A kit for characterising a target polynucleotide comprising (a) an analysis apparatus for characterising target polynucleotides and (b) a RecD helicase.