Colworth Medal Lecture - Biochemical Society Transactions

Superfamily I helicases as modular components of DNA-processing machines Mark S. Dillingham1 DNA:Protein Interactions Unit, School of Biochemistry, Faculty of Medical and Veterinary Sciences, University of Bristol, University Walk, Bristol BS8 1TD, U.K.

Delivered at the Edge, University of Sheffield, on 13 September 2010, as part of NACON VIII Mark Dillingham

Abstract Helicases are a ubiquitous and abundant group of motor proteins that couple NTP binding and hydrolysis to processive unwinding of nucleic acids. By targeting this activity to a wide range of specific substrates, and by coupling it with other catalytic functionality, helicases fulfil diverse roles in virtually all aspects of nucleic acid metabolism. The present review takes a look back at our efforts to elucidate the molecular mechanisms of UvrD-like DNA helicases. Using these well-studied enzymes as examples, we also discuss how helicases are programmed by interactions with partner proteins to participate in specific cellular functions.

Introduction A DNA helicase is an enzyme that catalyses the net conversion of dsDNA (double-stranded DNA) into ssDNA (single-stranded DNA), a key intermediate in many DNA transactions (Figure 1). Consequently, helicases play roles in virtually all aspects of DNA metabolism, including replication, repair and recombination (for general reviews, see [1–5]). The helicase reaction is thermodynamically unfavourable and is coupled to the hydrolysis of NTPs. Furthermore, the ssDNA products are either immediately handed on to other enzymes (e.g. polymerases) for further Key words: DNA recombination, DNA repair, PcrA, superfamily I helicase, UvrD. Abbreviations used: dsDNA, double-stranded DNA; SF, superfamily; ssDNA, single-stranded DNA; SSB, ssDNA-binding protein. 1 e-mail: [email protected]

Biochem. Soc. Trans. (2011) 39, 413–423; doi:10.1042/BST0390413

processing, or require stabilization by protein cofactors such as SSB (ssDNA-binding protein) in order to prevent spontaneous re-annealing. The reader should be aware that the meaning of the term ‘helicase’ has become somewhat blurred in the literature in recent years. Bona fide helicases (i.e. enzymes that catalyse the reaction depicted in Figure 1) are a subclass of a much larger collection of NTPases that were described by Gorbalenya and Koonin in 1993 [6] on the basis of primary structure similarities, and which constitute as much as ∼2 % of the proteome [7]. These were (and still often are) referred to collectively as ‘helicases’, despite the fact that it has subsequently become apparent that they do not all catalyse nucleic acid strand separation. Presumably, they do all share some core functionality, and this had been suspected to be ATP-dependent directional translocation along nucleic acids [8]. However, a current view of this ‘helicase-like’ enzyme class sees them minimally as energy-transducing machines, which exploit a common structural architecture to convert nucleotide binding and hydrolysis into mechanical work via protein conformational changes. The cyclic conformational changes generally result in the manipulation, translocation or unwinding of nucleic acids, but there are even examples of enzymes that act on peptide substrates [9]. The helicase-like enzymes are also distantly related to classical motor proteins and energytransducing systems including myosins and small GTPases. Indeed, certain principles behind their operation, such as the mechanism for ATP hydrolysis and the structural mechanism for localized propagation of conformational changes, are similar. The original bioinformatics analyses by Gorbalenya and Koonin [6] identified several SFs (superfamilies) of these NTPases that were defined by related (but different) sets of conserved amino acid motifs, and this classification was later expanded to include nucleic acid motors that belong to the AAA+ (ATPases associated with various cellular activities) family of ATPases [2]. In the present article, I will only be concerned with members of SF1, a group which includes the very intensively studied UvrD-like helicases, a particular focus of the present review. SF1 enzymes are distinguished by a particular set of conserved motifs that are broadly similar to, but easily distinguished from, those found in their close C The

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Figure 1 DNA helicase activity A DNA helicase (red triangle) catalyses the separation of duplex DNA into its component single strands in a reaction coupled to the hydrolysis of multiple ATP molecules. Purified helicases typically require an ssDNA tail flanking the duplex region of the substrate to act as a loading site, and move along ssDNA with a defined polarity. The enzyme depicted in this cartoon is a 3 →5 (or SF1A) enzyme and tracks along the grey DNA strand, displacing the orange strand. Many SF1 helicases are activated by interactions with partner proteins (blue star), although the mechanisms by which their unwinding activities are stimulated are not well understood. The ssDNA products of helicase may be stabilized and prevented from re-annealing by the binding of SSB or related proteins (green circles).

cousins from SF2 (for details, see [6,10,11]). To the best of our knowledge, all members of SF1 that have been studied in detail are true helicases, having been shown to catalyse ATP-dependent DNA (or RNA) strand separation in vitro. Whether or not they all do, and whether that activity is necessarily relevant in vivo are open questions. Nevertheless, we will concentrate here on bona fide unwinding enzymes. Much current and early work on helicase mechanism has employed a reductionist approach in which individual helicase proteins are overexpressed, purified and characterized using biochemical and biophysical methods. As will become apparent in the following sections, studies of this type have provided many key insights into the mechanism of UvrDlike enzymes. However, I will later argue that experiments on isolated helicases should be interpreted with caution, because many enzymes act in conjunction with protein partners and protein cofactors that activate and/or modify their activity (Figure 1).

General structure of UvrD-like SF1 DNA helicases The first crystal structure solved for any helicase was that of PcrA: an SF1 UvrD-like enzyme from Geobacillus stearothermophilus [12]. This was followed by several further structures of PcrA and other UvrD-like helicases, some of which were in complex with both DNA substrates and nucleotide analogues (Figures 2 and 3) [13–15]. All of the UvrD-like helicase structures determined so far share a foursubdomain structure exemplified by the structure of PcrA in complex with a DNA substrate and a non-hydrolysable ATP analogue (Figure 2A). The 1A and 2A subdomains form the helicase ‘core domains’ that are present in all C The

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SF1 and SF2 enzymes and function as the DNA motor [2]. Many of the conserved helicase motifs are found at the interface between the core domains, forming a single ATP-binding pocket (Figure 2B). An ssDNA-binding site extends across the top surface of both core domains, with the 3 -end on the 1A domain side. Several nucleobases of the ssDNA bind in pockets on each of the core domains via aromatic stacking interactions. The occupancy of these pockets is different depending on the status of the ATPbinding pocket and suggests a mechanism for directional translocation along ssDNA (see below). Two ‘accessory’ domains (1B and 2B) are inserts within the core domains and contact the duplex portion of the DNA substrate. We performed a comprehensive structure-based mutagenesis programme on PcrA, which, together with data from related enzymes, identified specific roles for the conserved helicase motifs in the DNA-unwinding reaction [10,14,16– 22]. The results are presented in a generalized form in Figure 2(C). Residues in motifs I, II and IV are involved in binding ATP and promoting its hydrolysis (coloured red); residues in motifs III and VI are γ -phosphate sensors involved in coupling ATP binding/hydrolysis to protein conformational changes required for DNA unwinding (coloured green) and residues in motifs Ia, 1b, III, IVa and V bind DNA (coloured blue). Several residues within the conserved motifs form interdomain contacts (coloured magenta) that probably mediate conformational changes of the core domains that lead to DNA translocation. Residues from outside the helicase motifs in accessory domains 1B and 2B are also involved in binding DNA, in particular with the duplex portion of the substrate. The structures of UvrD-like helicases have provided an important framework for the interpretation of biochemical and biophysical analyses.

Mechanism for directional translocation along DNA The motor mechanism which couples ATP hydrolysis to directional movement along DNA has long-occupied researchers in the field. In fact, it was not originally clear that the enzymes moved processively along DNA at all, and ‘distributive’ mechanisms (in which multiple helicases bound along a DNA length and caused local unwinding) were also discussed [23]. An important question had arisen from the simple observation that many helicases can only unwind DNA duplexes that are flanked by ssDNA of a particular polarity. If such helicases required a 5 -terminated ssDNA overhang, they were referred to as 5 →3 helicases, whereas those that required a 3 -terminated overhang (such as PcrA) were classified as 3 →5 helicases (as shown in Figure 1). Because duplex DNA is a quasi-symmetrical structure, this preference could arise either as a result of the asymmetric structure of the ss/dsDNA junction or because of the polarity of the flanking ssDNA [3]. In the latter case, unidirectional translocation along ssDNA in either the 3 →5 or 5 →3 direction could provide a simple explanation for the observed

Superfamily I helicases

Figure 2 Structure of PcrA helicase bound to DNA and AMP-PNP (adenosine 5 -[β,γ -imido]triphosphate) (A) Ribbon representation of the PcrA–DNA–AMP-PNP complex (PDB code 3PJR [15]) colour-coded and labelled according to the domain structure. The helicase core domains are shown in red and blue with AMP-PNP (pink) bound at their interface. The DNA substrate consists of a 10-bp duplex with a seven-base ssDNA 3 -overhang. The DNA is shown in black with the 3 -OH terminus of the ssDNA overhang indicated. The black arrow shows the direction of movement of the ssDNA relative to protein upon ATP-dependent translocation. (B) Structure of PcrA with the conserved amino acid motifs labelled according to the key shown to the right of the structure. The sequences of the motifs are displayed in WebLogo format [115] and were determined from a multiple alignment of the 500 sequences most similar to B. subtilis PcrA using BLAST [116] and COBALT [117]. Individual amino acids in the WebLogo motifs are colour-coded according to their function as assigned based on this crystal structure shown and extensive mutagenesis studies (red, ATP-binding/hydrolysis; green, γ -phosphate sensor; magenta, inter-core domain contact; blue, DNA binding; see the text for details). (C) Structure of PcrA with selected amino acids shown in stick representation. Amino acids are colour-coded according to their function using the same scheme as in the WebLogo motifs to the left of the structure.

Figure 3 Similar structures of UvrD-like helicases (A) Primary structure diagrams for all of the UvrD-like helicases found in two model bacterial organisms (E. coli and B. subtilis) as shown in Table 1. The subdomains are colour-coded according to the predicted or actual domain organization using the same scheme as for Figure 2(A). The four canonical subdomains (1A, 2A, 1B and 2B) are labelled in UvrD only, whereas additional domains are labelled for every helicase. The size of each domain is approximately to scale. Nt, N-terminus; Ct, C-terminus. (B) Comparison of the crystal structure of PcrA (G. stearothermophilus) with those of the E. coli helicases UvrD (PDB code 2IS4 [14]), Rep (PDB code 1UAA [13]) and RecB (PDB code 1W36 [52]). Domains are colour-coded using the same scheme as in Figure 2(A).

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unwinding polarity. Evidence for directional tracking along ssDNA by a DNA helicase had been provided by a study of the SF2 PriA helicase [24] and several groups had also inferred unidirectional translocation from the ssDNA lengthdependence of the ATPase activity [25–28]. However, results from experiments which investigated the effects of placing physical blocks in the path of the translocating helicase had seemed to exclude DNA tracking, instead suggesting a looping mechanism for translocation [29]. A looping mode for movement along ssDNA was a feature of the ‘active rolling model’, which, at that time, was a prominent putative mechanism for helicase activity [3,30]. It invoked a onedimensional random walk on ssDNA for translocation, with the unwinding polarity enforced by the structure of the ss/ dsDNA junction. A major step forward came with the development of the ‘streptavidin displacement assay’ by Morris and Raney [31]. Their work demonstrated that several different helicases were able to generate significant force against a streptavidin molecule bound to a biotin-modified oligonucleotide, provided that it was flanked by ssDNA of a particular polarity. This strongly suggested that the enzymes in question were coupling ATP hydrolysis to unidirectional translocation along ssDNA. The assay remains a simple and powerful means to investigate ssDNA translocation polarity that avoids the potential for equivocal results in a classical helicase assay caused by heterogeneous binding modes to the DNA substrate. However, it is unable to provide more quantitative information for tracking activity, such as the rate of movement or the relationship between ATP turnover and distance moved. Serendipitously, we developed two assays that overcame these limitations. Working with our PcrA mutants to probe the chemical mechanism of ATP hydrolysis, we had been investigating ATP turnover under pre-steady-state conditions using a biosensor for inorganic phosphate [17,32]. The ATPase activity of SF1 helicases is strongly stimulated by ssDNA, and so we employed a stopped-flow instrument to mix a pre-formed PcrA–ssDNA complex with ATP in the presence of the phosphate sensor. We observed a rapid release of phosphate followed by a slower steady-state turnover of ATP. Remarkably, the rapid phase of phosphate release was the result of several rapid turnovers of the ATPase and its amplitude was shown to be linearly dependent upon the length of the ssDNA. This relationship did not provide any insight into translocation polarity, but was qualitatively inconsistent with the active rolling model, because it showed that PcrA was moving unidirectionally along ssDNA. Importantly, it also provided one of the first estimates of the ‘ATP step size’, suggesting that one ATP molecule was required for every base translocated. Building on this work, we devised an assay to measure unidirectional ssDNA translocation directly and in real time [33]. Oligonucleotides were modified with the environmentally sensitive fluorescent base analogue 2-aminopurine at either the 3 - or the 5 -end to act as a proximity sensor for PcrA. This method confirmed that translocation was unidirectional and showed that it occurred in the 3 →5 direction at approx. 80 bases/s. C The


Lohman and co-workers have further developed the general methods for the analysis of ssDNA translocation kinetics [34–36]. Their work on the UvrD helicase also shows a step size of one ATP per base translocated, but provides evidence for an additional rate-limiting step that occurs on average every four or five bases. Recent studies using state-of-the-art single-molecule methods have provided interesting novel insights into the translocation properties of purified SF1 helicases [37]. For example, using TIRFM (total internal reflection fluorescence microscopy), the Ha laboratory has shown that UvrD-like helicases display a phenomenon termed ‘repetitive shuttling’ [38,39]. The enzymes undergo repeated cycles of 3 →5 tracking along ssDNA in single base steps, followed by a ‘snap-back’ towards the 3 -end. Such behaviour may be relevant to an anti-recombination function involving the removal of proteins such as RecA at replication forks by a ‘wire-stripping’ mechanism [39]. A magnetic tweezers approach was applied to the UvrD helicase [40,41]. Following tracking (and unwinding) into a duplex region of DNA, the enzyme spontaneously arrested translocation, switched DNA strands and translocated back towards the starting position, and it has been argued that such behaviour may also facilitate the removal of proteins from DNA [41]. Several studies with SF1 helicases have revealed kinetic disorder and/or pausing during translocation [39,42–47]. Interestingly, for the RecBCD complex, a pause occurs upon encounter with a specific regulatory sequence, χ [45], and this helps to enforce a final cleavage event on one strand of the nascent ssDNA to promote recombination at χ hotspot sequences. A molecular view of the process of ATP-dependent ssDNA translocation in single base steps has been provided by crystal structures of PcrA and UvrD with different nucleotide analogues [14,15]. These snapshots of the translocating helicase suggest that cycles of ATP binding and hydrolysis remodel the interaction between the core domains and the ssDNA, leading to the net movement of one base for each ATP hydrolysed in agreement with the biochemical data (reviewed in [2,48]). The mechanism has been likened to the movement of an inchworm along a twig, with the 2A domain acting as the head and the 1A domain as the tail, and its general feasibility is supported by molecular dynamics simulations [49,50]. PcrA and UvrD are examples of 3 →5 ssDNA motors and are classified as SF1A helicases. However, there are examples of SF1 enzymes that move in the 5 →3 direction (Dda and RecD are well-studied examples), and these are members of the SF1B class. Interestingly, a relationship between primary structure and translocation polarity is evident from a careful examination of the helicase motifs, and suggests that differences in residues in motifs Ia, Ib and 3 may be predictive of the polarity of a SF1 helicase [2,51]. Structures of the SF1B enzyme RecD2 in complex with ssDNA and ATP analogues indicate that these helicases track along ssDNA using an inchworm mechanism that is broadly similar to that employed by SF1A enzymes [51]. The origin of their different translocation polarities relates to the actual directionality of movement of the helicase relative to


ssDNA, rather than to the polarity with which ssDNA binds to the core domains. In other words, in an SF1A helicase the head of the inchworm is the 2A core domain, whereas the 2A domain acts as the inchworm’s tail in a SF1B helicase [51,52]. For SF1 helicases, there is now an emerging consensus in the field that the mechanistic basis for unwinding polarity relates to unidirectional ssDNA translocation at one base per ATP. However, there remains controversy over how this tracking activity is harnessed to promote processive duplex unwinding. It is perhaps important to point out at this stage that, in some cases, only the ssDNA translocation activity may be relevant to the in vivo function, as has been argued for the putative role of UvrD/PcrA in clearing RecA filaments from DNA [39,53,54].

Mechanism for separation of duplex DNA Lohman and Bjornson [3] differentiated between two forms of unwinding which were described as ‘passive’ and ‘active’. Although the process is always active in the traditional sense (ATP hydrolysis is absolutely required for unwinding), here the term is used to describe unwinding mechanisms in which the free energy of ATP binding and/or hydrolysis is used directly to destabilize the duplex. In contrast, passive unwinding mechanisms use energy from ATP in order to move along ssDNA, but only invade regions of duplex when nascent ssDNA arises spontaneously due to thermal fraying. The definitions of active and passive unwinding were formalized through the development of theoretical models for helicase action [55]. Subsequently, it was suggested that active and passive mechanisms might be best differentiated by comparing the rate of translocation along ssDNA with that for translocation through duplex DNA and, on this basis, UvrDlike helicases are active enzymes [56]. Unwinding models based on crystal structures of PcrA envisioned a relatively simple relationship between ssDNA translocation and duplex separation [15]. This involved an active melting of the duplex by the 2B accessory domain, which assisted the progress of the translocation motor into the duplex region of the substrate. This model was supported by mutagenesis studies which showed that point mutations in the DNA-binding residues of domain 2B reduced the observed helicase activity [19]. Similarly, the crystal structures of UvrD showed how the accessory domains ratcheted and twisted the duplex towards the ssDNA motor [14]. Paradoxically, however, the complete removal of the 2B domain in Rep helicase actually increases the observed helicase activity [57]. Moreover, the structural models invoke a monomer as the active form of the enzyme, and are at odds with biochemical observations that suggest a requirement for multiple helicase protomers to unwind DNA efficiently. Transient kinetics and single-molecule analyses of the PcrA, Rep and UvrD helicases have shown convincingly that monomers of these enzymes are unable to separate DNA duplexes under single turnover conditions [41,58–63]. By investigating the dependence of the unwinding reaction on the ssDNA tail length and the concentration of the helicase, it was shown that at least two helicase polypeptides are required

to observe DNA strand separation. However, there is as yet no convincing evidence that protein–protein interactions between the helicase protomers are needed for the enhanced unwinding. It therefore remains unclear whether the observed functional co-operativity simply reflects the combined activity of multiple autonomous monomers or whether physical association in a dimeric or multimeric helicase structure is required. Further work will be required to resolve the apparent differences between structure-based mechanisms for UvrD-like helicases and biochemical data for DNA unwinding. A possible explanation for the discrepancy may lie in the fact that many (and possibly all) UvrD-like helicases are dramatically stimulated by partner proteins or protein cofactors that are generally absent from helicase assays performed in vitro.

Activation of UvrD-like helicases by protein interaction partners and SSBs Within the cell, UvrD-like helicases rarely, if ever, function in isolation. Instead they are transient or stable components of larger molecular machines that catalyse a wide range of DNA-processing tasks. Table 1 shows the complete complement of UvrD-like helicases from a model Gramnegative (Escherichia coli) and Gram-positive (Bacillus subtilis) bacterium alongside their known functions. Each species contains four UvrD-like helicases, each of which has a specific cellular role, and some of which display multiple distinct functions. For each of these helicases that has been studied in significant detail, one or more interaction partners have been identified (Table 1). Given the similar underlying core structure of UvrD-like enzymes (Figure 3), it follows that functional specialization probably reflects specific properties of their accessory domains and/or partner proteins, which appropriately target or catalytically modify the helicase activity for the job at hand. In E. coli, the UvrD helicase itself is a key player in both the nucleotide excision repair and mismatch repair pathways [64,65]. Accordingly, cells from which UvrD is absent or in which UvrD is mutated are sensitive to DNA-damaging agents and UV light [66], but are also hyper-recombinogenic [67,68], suggesting a role in the suppression of illegitimate recombination. Indeed, UvrD has been shown to disassemble RecA nucleoprotein filaments in vitro [54], and this process may act at stalled replication forks to counteract deleterious activities of RecQ and RecFOR [69–71]. The closely related Rep helicase is essential for phage DNA replication and for efficient progression of the genomic DNA replication fork. Rep is required for replication restart at stalled forks directed by functional interactions with PriC [72]. Both Rep and UvrD also act as ‘accessory helicases’ that help to promote progression of the replication fork past protein roadblocks, especially the transcription apparatus [73–77]. In common with UvrD, the B. subtilis PcrA helicase is involved in UV repair, counteracts a toxic effect of RecFOR at replication forks [78,79], and inhibits RecA-dependent strand exchange in vitro [80]. Unlike either E. coli Rep or C The


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Table 1 UvrD-like SF1 helicases in E. coli and B. subtilis The protein partners column includes physical and functional interaction partners which are shown in-line with the relevant cellular function and key references.

Helicase

Organism

UvrD similarity (BLAST E value)

Biochemical activities

Cellular functions

Protein partners

Reference(s)

UvrD

E. coli

n/a

3 →5 helicase

Nucleotide excision repair Mismatch repair

UvrAB/UvrB MutL

[92,108] [85,94]

Anti-recombinase Accessory replicative helicase

RecA –

[54] [75,76]

Plasmid replication – Accessory replicative

– UvrD*† DnaB

[81] [62] [75,76]

helicase Phage replication

CisA

[109]

rpL14* Rep*† –

[87] [60] [110]

Rep

E. coli

10 − 103

3 →5 helicase

E. coli

2×10 − 21

3 →5 helicase

– – Recombinational repair

RecB

E. coli

10 − 6

[111]

B. subtilis

10 − 146

Double-stranded DNA break repair Plasmid replication

RecC and RecD

PcrA

3 →5 helicase–ssDNA endonuclease 3 →5 helicase

RepD

[82]

Nucleotide excision repair – –

– YxaL* rpL3*

[79] [84] [86]

–

RNA polymerase β-subunit* PcrA*†

[112]

Unknown Double-stranded DNA break repair

– AddB

– [113]

Unknown

–

[114]

HelD

– YjcD AddA

B. subtilis B. subtilis

4×10 − 69 6×10 − 21

Unknown 3 →5 helicase–ssDNA endonuclease

Possible 5 →3 helicase‡ *The significance (if any) of these interactions in vivo is not understood. YvgS

B. subtilis

10 − 5

[58,63]

†UvrD, Rep and PcrA show co-operativity in unwinding DNA in vitro, consistent with dimerization or higher-order self-interactions (see the text). ‡A YvgS-like protein from Deinococcus radiodurans displays 5 →3 helicase activity [114].

UvrD, the PcrA helicase is essential in B. subtilis and can rescue the non-viability of rep/uvrd double mutants of E. coli. This possibly reflects the partially redundant roles of Rep and UvrD as accessory helicases in E. coli being fulfilled by PcrA alone in B. subtilis [79]. The functional promiscuity displayed by PcrA, Rep and UvrD is further exemplified by their interactions with plasmid- and phage-encoded replication initiator proteins for rolling-circle replication of DNA [81–83]. Thus one helicase can operate in different pathways by assembly with different partner proteins. A common hallmark of helicase-partner proteins is their ability to stimulate the apparent helicase activity in vitro [72,82– 87]. For example, the PcrA helicase is comfortably able to unwind plasmid-sized DNA substrates in the presence of the replication initiator protein RepD [88,89]. Furthermore, many helicases require SSB [or other SSBs such as RPA (replication protein A)] as an additional protein cofactor to stabilize DNA-unwinding intermediates or products (Figure 1). For example, the rapid and processive unwinding C The


activities displayed by the AdnAB, AddAB and PcrA:RepD systems all require SSB ([89,90], and J.T.P. Yeeles, K. van Aelst, M.S. Dillingham and F. Moreno-Herrero, unpublished work). The precise mechanisms by which SSB-like proteins stimulate helicase activity are poorly defined and might vary between different helicases. Nevertheless, helicase assays are typically performed in the complete absence of SSBlike proteins or activator proteins and might therefore be misrepresentative of the catalytic potential of the enzyme under study. What do we know about how the UvrD-like helicases assemble with other cellular machinery to operate in different DNA-processing pathways? The Rep helicase is programmed for its role as an accessory helicase by targeting to the replisome via an interaction with the replicative helicase DnaB [76]. This interaction requires the C-terminal extension of Rep which, interestingly, is also the main region of Rep which differentiates it from the UvrD protein (Figure 3). The equivalent C-terminal region of UvrD is distinctive, and


has been shown to be important for dimerization and for interaction with the MutL, UvrB and RecA proteins [91–94]. These short C-terminal extensions are largely dispensable for helicase activity and seem to be key sites for protein interactions, but they have never been resolved in any crystal structure and may be natively disordered [13–15,92]. The 2B accessory domain may also play an important regulatory role in these enzymes as it has been shown to autoinhibit the Rep helicase [57]. It was suggested that Rep self-association or association with a partner protein may relieve this inhibition to activate DNA unwinding [1]. Furthermore, the 2B domain of a mycobacterial UvrD-like helicase was shown to be important for interaction with the Ku homodimer [95]. Interestingly, the 2B accessory domain is predicted to be missing (or very small) in some UvrD-like helicases such as HelD and YvgS (Figure 3). It has become important to develop a more sophisticated understanding of the molecular interactions between helicases and partner proteins and the functional and mechanistic consequences of such interactions. With these goals in mind, we have recently focused our attention on bacterial helicase–nucleases, such as RecBCD and AddAB, that are involved in the initiation of double-stranded DNA break repair by homologous recombination (Table 1) [96,97]. These stable multimeric complexes serve as model systems for understanding how a UvrD-like helicase (e.g. RecB) operates within a more physiological context (e.g. the RecBCD holoenzyme). The N-terminal region of the RecB polypeptide displays the four-subdomain structure that is typical of a UvrD-like helicase and a C-terminal extension forms a globular ssDNA endonuclease domain (Figure 3). As might be expected, the purified N-terminal helicase domain of RecB is (like other UvrD-like enzymes) a weak 3 →5 helicase [98]. In contrast, the RecBCD complex is an exceptionally powerful unwinding enzyme in vitro. It binds extremely tightly to free DNA ends and, in the presence of ATP, translocates along and unwinds duplex DNA at rates of up to 2000 bp/s and with an average processivity of approx. 30 kb, even in the absence of SSB [99–101]. As the enzyme moves along DNA, it occasionally cleaves both nascent ssDNA strands. Even more remarkably, the moving enzyme is able to scan the DNA for the recombination hotspot sequence χ . Upon recognition of this octameric ssDNA sequence, RecBCD briefly pauses before translocation resumes without accompanying cleavage of the 3 -terminated strand in order to promote RecA-dependent recombination [45,102,103]. In the context of the RecBCD complex, the RecB helicase is loaded on to its physiological substrate (a DNA end), is highly active (catalysing the fastest and most processive DNA unwinding reaction reported to date), and is coupled to other catalytic and DNA-binding activities. Part of the complexity of this system was explained when we and others demonstrated that RecBCD enzyme actually contains an additional motor domain [104,105]. The RecD subunit is also a DNA helicase, but displays a 5 →3 polarity. By engaging with either strand of the antiparallel DNA duplex, these two motor proteins could co-operate and drive the movement along the

DNA substrate using a ‘bipolar’ translocation mechanism. Interestingly, if either motor is inactivated by mutagenesis, the rate and processivity of the DNA translocation and unwinding is reduced [101]. Nevertheless, these single-motor variants of the RecBCD complex are still far more potent as helicases than isolated SF1 helicases such as Rep, UvrD or PcrA, and demonstrate clearly that a single helicase protomer is sufficient for rapid and processive DNA unwinding. Likewise, the functionally analogous AddAB-type helicase– nuclease catalyses rapid and processive DNA unwinding and does so powered by a single UvrD-like helicase motor [106]. The crystal structure of the RecBCD complex showed in detail how the RecB motor was integrated with other parts of the RecBCD machinery [52] (Figure 4A). The 2B accessory domain is the primary site for interaction with RecC protein, whereas domain 1B forms an ‘arm’ structure that contacts duplex DNA ahead of the translocating enzyme. The 1A and 2A core domains are engaged with the 3 strand of the duplex in essentially the same manner observed for PcrA, Rep and UvrD. The second motor (RecD) is poised to accept the 5 -strand of the duplex, such that the combined activities of the two bipolar motors can act in a complementary fashion as had been envisioned by the bipolar translocation model (Figure 4B). The combined activities of these motors pump each of the two single strands of DNA through enclosed tunnels within the complex, explaining how RecBCD is able to promote such highly processive translocation and unwinding. The tunnels lead to distinct exit points on the surface, which are both in close proximity to the RecB nuclease domain. The physical coupling of the two DNA motors to the nuclease domain allows both strands to be cut in a processive fashion by a single active site. Down-regulation of the cleavage of the 3 -strand is thought to occur when the recombination hotspot is recognized and sequestered by a χ -scanning device in the N-terminal region of RecC. Remarkably, the putative χ -binding site is structurally equivalent to the core domains of a UvrDlike helicase, but none of the characteristic helicase signature motifs are present (Figure 4B). It therefore appears that Nature has developed a sequence-specific ssDNA-scanning and -recognition device by modifying the ssDNA-tracking motor of a conventional helicase, providing a further example of the remarkable functional plasticity of the helicase-like protein architecture. From the RecBCD system, we learn an important lesson. If we were interested in the properties of the product of the recB gene, we might take the classic approach of cloning the gene, expressing and purifying the RecB protein and studying its properties in isolation. Indeed, such experiments have shown that the purified protein possesses modular 3 →5 helicase and endonuclease activities [98,107]. However, we would be unlikely to reach any useful conclusions about the complex biological role of RecB within the context of the RecBCD holoenzyme, because the complete machine is more than the sum of its parts. There are clearly limits to the utility of reductionist methods when studying a class of enzyme C The


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Figure 4 RecB and RecD helicases as components of the RecBCD helicase–nuclease (A) Crystal structure of the RecBCD complex (PDB code 1W36 [52]) shown in a ribbon representation. The RecB subunit is a UvrD-like helicase and has been highlighted by colour-coding according to the key shown above the structure. RecC and RecD are shown in light and dark grey respectively, and DNA is shown in grey (3 -terminated strand) and orange (5 -terminated strand). The 1A and 2A core domains are engaged with the 3 -terminated DNA strand in a similar manner to that observed for other UvrD-like helicases. The 1B accessory domain (yellow) forms an ‘arm’ structure that contacts duplex DNA, whereas the 2B accessory domain (green) forms a major protein–protein interaction site with RecC. (B) The RecBCD complex is shown in a similar orientation to (A), but is depicted as a ball-and-stick model with a grey surface. The protein has been cut back to reveal the channels for each ssDNA strand (highlighted in yellow) that pass right through the complex from the duplex binding site at the front, to the ssDNA exit points near the RecB nuclease domain at the rear of the complex. The SF1 helicase core domains (1A and 2A) of RecB and RecD are shown in red and blue as in other Figures. Note how the core domains of each helicase will engage with either strand of the antiparallel duplex to promote bipolar DNA translocation (see the text). The ‘helicase-like’ core domains of the RecC subunit are also indicated using the same colour scheme. These core domains are not thought to function as a translocation motor, but rather as a scanning device for the recombination hotspot sequence χ . Binding of χ to this part of the complex prevents the grey DNA strand from reaching the nuclease domain, thereby down-regulating cleavage of the 3 -terminated strand to promote recombination.

that is designed to act as a component of a multi-protein assembly.

Outlook It seems that the in vivo functions of UvrD-like helicases may be largely determined by their interaction partners and accessory domains. This observation raises many important fundamental questions regarding the structure, function and mechanism of this enzyme class. To what extent are the cellular functions of these enzymes degenerate or absolutely specific? Do UvrD-like helicases always function in the presence of partner proteins? What is the structural basis for, and what are the mechanistic consequences of, these interactions? To what extent are these interactions competitive, and can helicases be ‘re-programmed’ through protein design to perform novel tasks? Future experiments will address these questions by identifying and characterizing novel interaction partners, and by studying UvrD-like enzymes in the presence of protein partners or within the livecell context. It would be easy to fall into the trap of assuming that the lessons learnt from studies on SF1 DNA helicases to date (which have been heavily restricted to members of the UvrD- and Dda-like families), will necessarily apply across the whole SF. Research on the wider group of helicase C The


like ATPases has already shown them to be involved in an incredibly wide range of processes, acting not only as helicases, but also as DNA translocases and switches, and on substrates other than nucleic acids [2,4,5]. There is still much to learn about this remarkably diverse group of enzymes.

Acknowledgements I thank my mentors for their continued support and encouragement and for their contributions to much of the work described in this review: Steve Halford, Mark Szczelkun, Dale Wigley, Panos Soultanas, Steve Kowalczykowski and Martin Webb. I am indebted to past and present members of my own laboratory for their work and enthusiasm: Joe Yeeles, Emma Gwynn, Neville Gilhooly and Kara van Aelst.

Funding I am grateful to the Wellcome Trust, the Royal Society and the European Research Council for providing major funding for my research.


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