Review

Accessory Replicative Helicases and the Replication of Protein-Bound DNA

Jan-Gert Brüning, Jamieson L. Howard and Peter McGlynn Department of Biology, University of York, Wentworth Way, York YO10 5DD, United Kingdom

Correspondence to Peter McGlynn: [email protected] http://dx.doi.org/10.1016/j.jmb.2014.10.001 Edited by B. Connolly

Abstract Complete, accurate duplication of the genetic material is a prerequisite for successful cell division. Achieving this accuracy is challenging since there are many barriers to replication forks that may cause failure to complete genome duplication or result in possibly catastrophic corruption of the genetic code. One of the most important types of replicative barriers are proteins bound to the template DNA, especially transcription complexes. Removal of these barriers demands energy input not only to separate the DNA strands but also to disrupt multiple bonds between the protein and DNA. Replicative helicases that unwind the template DNA for polymerases at the fork can displace proteins bound to the template. However, even occasional failures in protein displacement by the replicative helicase could spell disaster. In such circumstances, failure to restart replication could result in incomplete genome duplication. Avoiding incomplete genome duplication via the repair and restart of blocked replication forks also challenges viability since the involvement of recombination enzymes is associated with the risk of genome rearrangements. Organisms have therefore evolved accessory replicative helicases that aid replication fork movement along protein-bound DNA. These helicases reduce the dangers associated with replication blockage by protein–DNA complexes, aiding clearance of blocks and resumption of replication by the same replisome thus circumventing the need for replication repair and restart. This review summarises recent work in bacteria and eukaryotes that has begun to delineate features of accessory replicative helicases and their importance in genome stability. © 2014 MRC Laboratory of Molecular Biology. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).

Introduction Replication forks break down in vivo with sufficient frequency to create major challenges to genome duplication [1,2], although there are still no robust estimates of exactly how frequently replisomes stall inside cells. However, it is clear from the phenotypes associated with mutations in replication repair enzymes that failure to resuscitate blocked replication forks results in genome rearrangements and cell death [3]. There are many potential sources of replication barrier in vivo. Damage to the template DNA, proteins bound to the template and non-B-form DNA structures all have the ability to halt replication forks either by inhibiting DNA polymerases or by preventing translocation of the replicative helicase. The relative importance of such barriers will depend to some extent on environmental factors such as

sources of DNA damage. It is becoming apparent, though, that high-affinity noncovalent nucleoprotein complexes, especially those associated with transcription, are a major threat to DNA replication in both bacteria and eukaryotes [4,5]. Indeed, protein– DNA complexes are the dominant source of replication fork pausing in Escherichia coli in the absence of exogenous DNA damaging agents [6]. Proteins can also form covalent bonds with DNA but the frequency of formation of such covalent adducts is unclear. Clearance of covalently bound protein–DNA complexes also requires different mechanisms to those needed for noncovalent nucleoprotein complexes [7] and thus will not be dealt with in this review. A replisome can halt at a noncovalent protein– DNA complex but if the nucleoprotein complex can be disrupted then the same replisome could continue translocation. However, if the replisome

0022-2836/© 2014 MRC Laboratory of Molecular Biology. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). J. Mol. Biol. (2014) 426, 3917–3928

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dissociates from the DNA prior to removal of the barrier then the replisome must be reloaded in order for replication to resume. Thus the term pausing must be emphasised. Replisome pausing can result in either resumption of replication by the same replisome or the need for blocked fork processing to load a new replisome back onto the chromosome. Work by several laboratories in recent years has demonstrated that a group of enzymes termed accessory replicative helicases facilitate disruption of nucleoprotein complexes ahead of the replication fork. In bacteria and probably in eukaryotes this disruption can occur in the presence of a paused but still active replisome to allow resumption of replication without the dangers of blocked fork processing and replisome reloading [6,8–13].

The problem with nucleoprotein complexes Unwinding of the template strands is a key feature of DNA replication, allowing access to the bases by DNA polymerases. However, proteins bound to the double-stranded template make multiple noncovalent bonds with the DNA that must also be disrupted to effect duplex unwinding. Translocases are molecular motors that couple the hydrolysis of nucleoside triphosphates, usually ATP, with movement along single- or double-stranded nucleic acid. Helicases are a type of nucleic acid translocase that couple this movement to unwinding of the strands within a nucleic acid duplex [14–16]. DNA unwinding at the replication fork is performed by hexameric helicases within the context of the replisome, with the hexameric rings encircling either the lagging or the leading strand template in bacteria and eukaryotes, respectively [17]. These “molecular wire strippers” translocate along the single-stranded DNA (ssDNA) to accomplish duplex unwinding [18] (Fig. 1). The number of base pairs unwound per ATP hydrolysed by these enzymes is still unclear [14], although for the bacterial replicative helicase, DnaB,

helicase movement

Fig. 1. Unwinding of DNA by replicative helicases. Replicative helicases are hexameric and encircle the DNA strand along which they translocate. Exclusion of the complementary DNA strand from the hexameric ring results in unwinding of the duplex.

there is evidence that two base pairs are unwound per ATP hydrolysed [19,20]. The free energy of hydrolysis of ATP is approximately 42 kJ/mol under physiological conditions whilst only approximately 6.7 kJ/mol is required to open a single base pair of DNA of average composition [21]. Disruption of both the interactions between the DNA strands and interactions between bound protein and the DNA is therefore thermodynamically possible. Displacement of proteins bound to DNA is also likely to be a multistep rather than a single step process, involving progressive disruption of intermolecular contacts [22] as the helicase proceeds. Thus disruption of a high-affinity protein–DNA interaction will likely involve hydrolysis of multiple ATP molecules, increasing the probability of nucleoprotein disruption. Indeed, some hexameric helicases can unwind duplex DNA that harbour high-affinity nucleoprotein complexes [23–25]. Even in situations where an isolated replicative helicase cannot efficiently unwind protein-bound duplex DNA, it must be borne in mind that such helicases function within the context of large macromolecular machines that have both a thermodynamic and a kinetic effect on helicase activity [17,21]. These interactions amongst the helicase, polymerases, ssDNA binding proteins and clamp loaders accelerate DNA unwinding and enhance processivity in bacteria, bacteriophages and mitochondria [26–29]. As an example, a single lac repressor–operator complex inhibits the E. coli replicative helicase DnaB in the absence of other components of the replisome [23] but this high-affinity protein–DNA complex is much less effective at inhibiting translocation of the E. coli replisome [30]. This enhanced ability to displace proteins from DNA may be related to whether unwinding by the helicase is active or passive. Active unwinding involves destabilisation of base pairing at the ssDNA–double-stranded DNA (dsDNA) junction by interaction with the helicase whilst passive unwinding relies more on thermal fluctuations to generate ssDNA that the helicase can subsequently trap by translocation [31–33]. It is difficult to envisage how a passive mechanism could lead to disruption of a nucleoprotein complex, given the much higher activation barriers for duplex opening when bound by proteins. As pointed out by Manosas et al., several hexameric replicative helicases display largely passive unwinding mechanisms in the absence of other replisome components with rates of DNA unwinding being much lower than rates of translocation [32]. The accelerated unwinding displayed by these helicases when part of a replisome indicates that more active unwinding mechanisms operate at replication forks, correlating with an increased propensity of these motors to translocate through nucleoprotein complexes. However, the physical basis of this enhanced protein displacement remains unclear. Indeed, very little is known

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about the mechanisms behind helicase-catalysed protein displacement from DNA. It is assumed that protein displacement and active DNA strand separation are achieved via the same ATP-driven mechanism but whether this is actually the case remains to be demonstrated. Proteins bound to DNA can be displaced by replicative helicases within the context of the replisome. However, replisomes must duplicate many kilobases or even megabases of DNA and thus will encounter a large number of nucleoprotein complexes. A low probability of a single high-affinity nucleoprotein complex blocking replisome movement will therefore translate into a high probability of such blockage occurring within the context of genome duplication. Dynamic heterogeneity driven by conformational fluctuation within individual enzymes [34,35] may also contribute to a distribution of probabilities concerning replication fork blockage by nucleoprotein complexes. A complicating issue is that loading of replicative helicases onto the chromosome is a tightly regulated process in all organisms since this is the key step in initiation of DNA replication. Thus replication forks in bacteria contain a single hexameric replicative helicase with no known means of loading additional hexamers and the same might also be true in eukaryotes. Why might this present problems? Multiple helicases can cooperate functionally to reduce inhibition of DNA unwinding by bound proteins [36,37] but the absence of multiple replicative helicases at a single fork precludes cooperative removal of protein–DNA barriers. What are the primary sources of nucleoprotein barriers inside cells? Transcribing RNA polymerases are very high affinity protein–DNA complexes present in high abundance on chromosomes and are powerful translocases in their own right [38]. Such complexes present major unavoidable barriers to genome duplication in both bacteria and eukaryotes, the details of which have been reviewed extensively elsewhere [4,5,39]. However, any high-affinity nucleoprotein complex presents challenges to replication fork movement, reflecting the largely nonspecific nature of these barriers [9,30,40].

ly, in a co-directional collision with a transcribing RNA polymerase, the replisome might continue translocation behind the moving RNA polymerase until a transcription terminator disengages the transcription complex from the chromosome. The halted replisome might also lose activity before dissociation of the block has occurred. Whether paused replisomes resume duplication or lose activity is determined by the stability of paused replisomes and the rate of spontaneous or induced dissociation of the blocking nucleoprotein complex. Reconstituted E. coli replisomes retain function for at least a few minutes regardless of the source of the block [41–44] providing a window of opportunity for removal of the replicative barrier. If this window passes, the pathways by which paused replisomes lose function are unknown but the result is that the replisome must be reassembled back onto the chromosome, a process that is important not only in bacteria but also in eukaryotes [2,45– 47]. Mechanisms that promote the resumption of replication by paused replisomes before they lose activity would avoid the risks to genome stability associated with replisome reassembly [3]. This promotion could be achieved by two general mechanisms that are not mutually exclusive. (1) Enhancing the stability of paused replisomes would increase the probability that the blocking protein will dissociate from the template before the replisome loses function, allowing replication to continue. (2) Accelerating the rate of dissociation of blocking nucleoprotein complexes could promote resumption of movement by a paused replisome. This acceleration could conceivably occur via the paused replisome itself if it continues to exert force on the nucleoprotein complex. Alternatively, other motor proteins could provide the force needed to induce dissociation of the protein–DNA barrier. There is extensive evidence that eukaryotes have specific mechanisms to stabilise blocked forks but such mechanisms appear absent in bacteria [2]. However, evidence that both bacteria and eukaryotes increase the rate of dissociation of blocking protein–DNA barriers is emerging.

Replication fork pausing and breakdown

Helicases and the replication of protein-bound DNA

An encounter between a replication fork and a nucleoprotein complex can result in several outcomes. The replisome itself could induce dissociation of the protein from the DNA and continue replicating the template with little, if any, impact on fork movement. Alternatively, the replisome might halt at the protein–DNA barrier but retain function and then continue replication after subsequent spontaneous or induced dissociation of the barrier. Similar-

The ability to disrupt proteins bound to DNA is a common property of helicases [23,36,37,48–50]. Helicases therefore provide a potential means to increase the rate of dissociation of protein–DNA replicative barriers. The first suggestion that accessory helicases might accelerate replication fork translocation along protein-bound DNA arose from the finding that DNA unwinding by E. coli Rep helicase was not substantially inhibited by a lac

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(a) 5’

(b)

E. coli 3’

S. cerevisiae 5’

3’

Rep

Mcm2-7

Protein block replisome

3’ 5’

Protein block 5’ 3’

DnaB

helicase movement

5’ 3’

replisome

Rrm3 3’ 5’

helicase movement

Fig. 2. Complementary translocation polarities by primary and accessory replicative helicases. (a) The replicative helicase DnaB translocates 5′-3′ along the lagging strand template in bacteria. The E. coli accessory replicative helicase Rep translocates with 3′-5′ polarity along the leading strand template. The physical interaction between Rep and DnaB is indicated. (b) In eukaryotes, the replicative helicase MCM2-7 translocates 3′-5′ along ssDNA whereas the known accessory replicative helicases translocate 5′-3′. Interaction of S. cerevisiae Rrm3 with components of the replisome is indicated.

repressor–operator complex in vitro whereas the replicative helicase DnaB was inhibited [23]. This model was built on (1) the hijacking of Rep as a replicative helicase by several bacteriophages [51]; (2) the viability of cells lacking Rep [52], hence excluding it as the primary replicative helicase; and (3) a reduced rate of E. coli chromosome replication in cells lacking Rep [52,53]. Moreover, cells lacking Rep are inviable in the absence of RecBCD [13], a helicase/exonuclease that degrades blunt dsDNA ends to generate ssDNA onto which the strand exchange protein RecA is loaded [54]. This requirement for repair of dsDNA ends was attributed to increased replication blockage in the absence of Rep [55,56]. The first direct evidence that helicases other than the hexameric replicative helicase play a critical role in duplicating protein-bound DNA came from Saccharomyces cerevisiae. Virginia Zakian and colleagues demonstrated that the S. cerevisiae Rrm3 helicase is required for normal rates of fork progression and the minimisation of fork blockage through nonhistone protein–DNA complexes across the yeast genome [9,57–59]. This helicase was associated with the replication fork via interactions with DNA polymerase ε and the sliding clamp, indicating that Rrm3 acts locally within the vicinity of the fork [60,61]. Yeast two-hybrid analysis also indicated that Rrm3 interacts with the origin recognition complex, suggesting that Rrm3 is loaded at a very early stage of S-phase [60,62]. Although cells lacking Rrm3 are viable [8], activation of the intra-S-phase checkpoint is important to maintain this viability indicating the importance of Rrm3 in minimising genome duplication problems [60]. Rrm3-deficient cells also display hyperrecombination [8], a hallmark of replicative defects [3], and depend on alternative fork processing pathways for survival [10,11,63].

Clearing a path ahead of the fork The above mentioned studies in yeast demonstrated unequivocally that, in addition to the main replicative helicase that drives strand separation at the fork, other helicases play important roles in replicating protein-bound DNA. Exactly what these roles were remained uncertain. This issue was resolved by the demonstration that Rep aids progression of a replisome through nucleoprotein complexes in vitro [12]. A homologous E. coli helicase, UvrD, also possesses this ability [12]. The known inviability of E. coli strains lacking both Rep and UvrD [64] could therefore be attributed to the absence of accessory replicative helicase activity that displaces blocking proteins ahead of the fork [12,65]. Transcription complexes were primarily responsible for the lethality of Δrep ΔuvrD cells, supporting the idea that high-affinity nucleoprotein complexes generated the need for accessory replicative motors [12,65,66]. Conflicts between replication and transcription are emerging as a key problem in the replication of all genomes [59,67–69].

Polarity All helicases demonstrated to accelerate fork movement along protein-bound DNA are members of helicase Superfamily 1, a class of helicases that translocate along ssDNA either with 3′-5′ or with 5′-3′ polarity [14–16]. These translocation properties imply that these helicases translocate along single-stranded template DNA at the fork to facilitate displacement of nucleoprotein barriers. A Superfamily 2 helicase, E. coli DinG, is also needed for efficient resolution of conflicts between replication and transcription that is attributable, at least in part,

Review: Accessory Replicati

to the ability of DinG to remove R-loops [65,70]. However, whether DinG can also disrupt nucleoprotein complexes ahead of replisomes is currently unclear. The polarity of translocation of these Superfamily 1 accessory helicases differs between bacteria and eukaryotes. Rep and UvrD translocate 3′-5′ [71,72] whereas Rrm3 translocates 5′-3′ along ssDNA [58]. These differences may be significant. The hexameric replicative helicases in bacteria and eukaryotes translocate along ssDNA with 5′-3′ and 3′-5′ polarity, respectively. Thus, in both E. coli and S. cerevisiae, the primary and accessory replicative helicases translocate along opposing template strands at the fork (Fig. 2). Such an arrangement might be dictated via occlusion of ssDNA by the hexameric replicative helicase at the DNA junction. Although speculative, this model is supported by the ability of a Bacillus 3′-5′ Superfamily 1 helicase, PcrA, to complement E. coli Δrep ΔuvrD lethality in vivo and promote E. coli fork movement through protein–DNA complexes in vitro [12,73]. In contrast, 5′-3′ Superfamily 1 helicases from Deinococcus radiodurans and bacteriophage T4 fail to do either [12]. Identification of the Schizosaccharomyces pombe 5′-3′ helicase Pfh1 as an accessory replicative helicase supports the importance of having two types of helicase with opposing polarities at the replication fork [68,69,74,75]. It is implicit in this model of complementary helicase polarities at the fork that accessory helicases access ssDNA on one of the template strands to allow translocation towards the nucleoprotein barrier. Rep makes critical contacts with five nucleotides of ssDNA [76]. Whether this amount of ssDNA is exposed on the leading strand template during active replisome movement or only upon replisome pausing is unknown.

Localisation The ability of the Bacillus 3′-5′ Superfamily 1 helicase PcrA to act as an accessory motor at the E. coli replication fork in vitro and in vivo [12,73] suggests no need for motor specificity other than that it translocates along ssDNA with a polarity opposite that of the primary replicative helicase. However, S. cerevisiae Rrm3 associates physically with the replication machinery (see above) [60,61]. The C-terminus of Rep interacts physically with the E. coli replicative helicase DnaB and this interaction plays an important role in promoting replication of protein-bound DNA [12,77]. These interactions appear at odds with the lack of specificity suggested above. However, a comparison between Rep and UvrD is informative. UvrD does not interact physically with components of the replisome and this lack of interaction correlates with the ability of UvrD to

3921 only partially compensate for the absence of Rep [6,12,77]. Rep, not UvrD, therefore acts as an accessory motor at the fork in wild-type cells and UvrD partially compensates for the absence of Rep by virtue of the high intracellular UvrD concentration [77,78]. This partial compensation by UvrD could be driven, at least in part, by a physical interaction between UvrD and RNA polymerase, positioning UvrD at the most important type of nucleoprotein replicative barrier [79,80]. It is unclear whether this interaction has evolved to facilitate UvrD accessory helicase activity since the functioning of UvrD in mismatch and nucleotide excision repair could also be facilitated by this UvrD–RNA polymerase interaction [80–82]. Moreover, the presence of Rep in E. coli would appear to obviate the need for UvrD to act as an accessory helicase under normal circumstances. However, Rep is restricted to γ proteobacteria. Bacillus species lack Rep and their likely accessory replicative helicase is PcrA, a close homologue of UvrD that also interacts with RNA polymerase [79]. There is also evidence that PcrA might interact with the ribosome [83]. The coupling of transcription and translation in bacteria might therefore provide another platform for locating accessory replicative helicases in the vicinity of common replicative barriers. There might therefore be two general localisation mechanisms for accessory replicative helicases in which these motors interact either with the replisome or with important types of nucleoprotein barrier. However, any high-affinity nucleoprotein complex can act as a replicative barrier. Localising the accessory replicative helicase at transcription complexes rather than the replisome would prevent acceleration of fork movement through protein–DNA complexes not associated with transcription. When an accessory helicase interacts physically with the replisome, it is still not clear whether such helicases are associated continuously or transiently with the replication fork. One possibility is that these helicases associate only when the replisome pauses at a barrier. Although chromatin immunoprecipitation indicates that Rrm3 is continuously associated with the fork [60], it remains possible that frequent replisome pausing gives the impression of continuous association. Likewise, the relatively high affinity of Rep for DnaB hints at continuous association but the equilibrium dissociation constant of less than 100 nM was estimated with Rep and DnaB in isolation in vitro [12]. It is possible that the Rep interaction domain within DnaB remains inaccessible within the context of the replisome except under certain circumstances such as replisome pausing. Even if an accessory helicase is continuously associated with the replisome, it is unclear whether the helicase is bound to, or translocating along, ssDNA. The unwinding rate of

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(a) 5’

(b)

5’

3’

(c)

5’

2B 1B

2A

5’

3’

5’

1A E. coli Rep

E. coli UvrD

B. stearothermophilus PcrA

Fig. 3. Conserved domain structure of Superfamily 1 helicases. Crystal structures of (a) E. coli Rep bound to ssDNA (PDB ID: 1UAA [76]), (b) E. coli UvrD bound to partial duplex DNA (PDB ID: 2IS2 [105]) and (c) Bacillus stearothermophilus PcrA bound to partial duplex DNA (PDB ID: 3PJR [103]) in cartoon representation. The conserved domain structure is illustrated by colour coding with the 1A subdomain in green, the 1B subdomain in yellow, the 2A subdomain in blue and the 2B subdomain in red. The arrow indicates the direction of movement for all three helicases in the 3′-5′ direction along the ssDNA bound between subdomains 1A and 2A.

the E. coli replisome is 246 bp/s at 23 °C [84] whilst Rep unwinds DNA at 57 bp/s at 25 °C [85]. This disparity in unwinding rates suggests that Rep does not contribute to template duplex separation at the fork. However, it remains possible that the unwinding rate of Rep when operating as part of the replisome is much higher than Rep alone. Alternatively, Rep might not unwind duplex DNA at the fork but might translocate along ssDNA that is being exposed transiently on the leading strand template, given the faster rate of ssDNA translocation by Rep as opposed to duplex DNA unwinding. Rep translocates at 279 nt/s at 25 °C [86], similar to the speed of the replisome [84].

Cooperativity The ability of Rep to interact with the replicative helicase in E. coli is reflected in cooperativity between Rep and DnaB in unwinding forked DNA in vitro [12,87]. In contrast, there is no observed cooperativity between UvrD and DnaB, which correlates with the lack of physical interaction between these two helicases [12]. This cooperativity could be explained by increased local concentrations of Rep at the fork via the Rep–DnaB interaction (see above). Monomers of Rep, UvrD and PcrA are unable to catalyse DNA strand separation in vitro in the absence of ssDNA binding proteins or partner proteins [85,88–93]. Increased local concentrations might therefore be required to ensure multiple monomer loading to allow efficient DNA strand separation and, by implication, protein displacement. However, the presence of not only SSB (single-stranded binding protein) but also DNA

polymerases at the fork might enhance monomeric helicase-catalysed strand separation by trapping the ssDNA produced by monomeric helicases [29,94,95]. Moreover, physical and functional coupling of Superfamily 1 helicases to other proteins can have significant impacts on motor activity as illustrated by the increased processivity of PcrA upon interaction with the plasmid initiator protein RepD [96]. Regardless of the need for multiple helicase molecules to effectively unwind duplex DNA, loading of multiple monomers facilitated by physical interactions with the replisome might be important for nucleoprotein disruption. Monomers of the bacteriophage Superfamily 1 5′-3′ helicase Dda can unwind dsDNA but multiple Dda molecules are needed to efficiently disrupt protein–DNA interactions [36,37,97]. This requirement for multiple Dda molecules does not appear to require specific protein–protein interactions but may be needed for increased force production and/or prevention of backwards slippage of the lead helicase molecule along the ssDNA [36,37]. Loading of multiple helicase monomers onto ssDNA implies that SSBs do not prevent such loading. E. coli SSB displays increasing dissociation rates as the length of ssDNA available for binding decreases below 35 nt [98,99]. Superfamily 1 helicases bind to 5–6 nucleotides of ssDNA [95]. Thus if only short stretches of ssDNA are exposed at the fork then it may be accessible to helicases, especially if local helicase concentration is elevated via physical interactions with the replisome. The ability of additional helicases to operate at the fork to displace protein–DNA complexes appears to circumvent the limitation of having only a

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(a) 5’

2B

1B 2A 1A

(b)

2B 1B 3’ 2A

(c)

2B (open)

1A

2B (closed)

Fig. 4. Conformational changes within Rep. Crystal structures of E. coli Rep in (a) the closed conformation and in (b) the open conformation of the 2B subdomain (PDB ID: 1UAA [76]). These two structures are related by a rotation of 130° around a hinge region connecting the 2B subdomain to the 2A subdomain. Colour coding is as in Fig. 3. (c) Overlay of both Rep conformations with the 1A, 1B and 2A subdomains in grey and the 2B subdomain in the open and closed conformation in red and blue, respectively.

single replicative helicase within the replisome. At least two motors can therefore be brought to bear on any protein roadblocks to replication. Moreover, Superfamily 1 helicases have limited processivity in the absence of other factors [100]. The activity of accessory replicative helicases might therefore be restricted to the vicinity of the fork, a potentially important consideration given the problems associated with unrestricted unwinding of DNA. However, whether this low processivity is also manifested

in accessory helicases when interacting with the replisome is unknown. It is also possible that, even if accessory helicase processivity is elevated within the context of the replisome, the very high translocation rates of replisomes might result in the fork rapidly overtaking any accessory motors. Regardless of any effects of local accessory helicase concentration, allostery via interactions between accessory helicases and other enzymes at the replication fork is also possible. Physical interactions between the Rep C-terminus and DnaB [12] and the large conformational transitions that Rep is known to undergo [76] make it tempting to speculate that allosteric interactions might be important in E. coli. Rep, UvrD and PcrA possess four subdomains, 1A, 1B, 2A and 2B (Fig. 3) with the 1A and 2A domains harbouring all of the conserved helicase signature motifs that constitute the translocation motor [15,95]. Crystal structures of a Rep– ssDNA complex indicate that the 2B domain can exist in “open” and “closed” conformations that differ by rotation of approximately 130° about a hinge region connecting it to the 2A domain [76] (Fig. 4). Switching between open and closed conformations has also been observed by FRET (fluorescence resonance energy transfer) as single molecules of Rep translocate along partial duplex DNA substrates [101], although the closed conformation may predominate [102]. However, the functional significance of the open and closed conformations remains unclear. The 2B domains of PcrA and UvrD interact with duplex DNA ahead of the ssDNA– dsDNA junction and assist in melting of the duplex ahead of the 1A/2A motor or wrenching the duplex into the motor [103–106]. However, it is unclear whether the Rep 2B subdomain also interacts with duplex DNA. Indeed, removal of the Rep 2B domain activates unwinding, suggesting that this domain has an autoinhibitory role [86,107]. Interactions with either other Rep monomers or different proteins might activate Rep helicase via a 2B conformational transition, potentially providing a means to switch on Rep activity when and where required [86,107]. Perhaps interaction with DnaB acts as such a conformational switch [12] although it is not obvious from the structure of Rep how such a switch would operate. The 2B subdomain might also be important for a different reason. Consideration of the structures of UvrD and PcrA bound to partial duplex substrates indicates that the 2B subdomain would be the initial point of direct contact between the helicase and any protein–DNA barrier (Fig. 3) [103,105]. The importance of such a contact for nucleoprotein disruption is not known but might have impact not only on the structure of the nucleoprotein complex but also on the structure of the helicase, especially given the conformational flexibility within these motor enzymes.

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When accessory helicases fail If translocation of the primary replicative helicase is inhibited by nucleoprotein complexes and accessory helicases fail to clear the blocking proteins, then the replisome will come to a halt. How frequently and under what circumstances such blockage occurs in wild-type cells is unclear. Protein–DNA complexes are the main sources of replisome pausing in E. coli but in the presence of Rep many of these pausing events do not lead to replisome inactivation [6]. However, failure of a single replisome can challenge viability and so even a very low frequency of failure may be significant. Indeed, the potential for arrays of transcribing RNA polymerases to form behind a blocked transcription complex might create nucleoprotein barriers that are difficult to overcome even with accessory replicative helicases being present at the fork [6,65,67]. How can genome duplication be completed successfully if a replisome encounters a protein– DNA barrier that cannot be overcome by helicases at the fork? The potential exists in eukaryotes for a converging fork to arrive from an adjacent replication origin thus avoiding the need to repair or reactivate the blocked replisome [108]. However, if a nucleoprotein barrier such as an array of RNA polymerases can block one fork then it might also be able to block a converging fork, leaving a portion of the genome unreplicated. Regions of the genome with a low density of origins or that are replicated unidirectionally would also be unable to rescue a blocked fork in a timely manner and such regions are often associated with increased risks of genome instability [109–111]. It might therefore be necessary to remove nucleoprotein blocks via alternative pathways that do not rely on accessory replicative helicases. There are many transcription elongation factors that facilitate the restart or dissociation of halted RNA polymerases [4,5]. If the paused replisome retains function for a sufficient length of time then clearance of a blocked transcription complex might allow resumption of replication by the same replisome. However, if the replisome loses function prior to clearance of the block then reloading of the replication machinery would become necessary. Much is known about replisome reloading mechanisms away from origins in bacteria and it is clear that such reloading also occurs in eukaryotes [2]. How might a reassembled replisome translocate successfully through a nucleoprotein block that has previously halted replication? One possibility is that, since such blockage is stochastic, a reassembled replisome might succeed in translocating through a block where the initial fork failed [30]. It is also possible that replisome reassembly extends the window of opportunity for the original nucleoprotein block to either dissociate

Review: Accessory Replicati

spontaneously or be actively removed, for instance, by transcription elongation factors. Replisome reloading does come at a cost. DNA processing is often required at inactivated replication forks and the involvement of recombination enzymes results in genome instability being an unavoidable price to pay for efficient replisome reloading [112–114]. This is illustrated by increased mitotic recombination in the absence of the accessory replicative helicase in S. cerevisiae [8]. Moreover, RecFOR-dependent loading of RecA contributes to the viability problems in E. coli lacking both Rep and UvrD [12,65,115]. The accessory helicase in S. pombe, Pfh1, is also required for the maintenance of genome integrity at stable protein– DNA complexes, raising the possibility that the human homologue, hPif1, performs a similar function [68,69]. Indeed, mutation of hPif1 in three high-risk breast cancer families might be at least partially explained by difficulties in replicating through nucleoprotein complexes [68,116]. However, hPif1 may also regulate telomerase and promote replication of G quadruplex structures in DNA, complicating interpretation of mutant phenotypes [117,118].

Concluding remarks We have learnt a great deal about accessory replicative helicases over the last decade. However, we still know very little about how interactions with the replisome alter accessory helicase activity. We also know nothing about how such interactions might alter replisome activity. For example, does interaction of Rep with DnaB alter the activity of one or both motors? More generally, whilst we have an increasing understanding of the physical basis of DNA strand separation catalysed by helicases, we know little about how these motor enzymes disrupt protein–DNA complexes. This requirement for nucleoprotein complex disruption is a ubiquitous feature of life, given that such complexes present replicative barriers in all organisms. The importance of accessory replicative helicases in human health is also only just beginning to be explored. These enzymes are likely to be critical not only in terms of maintaining genetic stability in human cells but also in facilitating infections by ensuring rapid cell division of bacterial pathogens.

Acknowledgements Work in the authors' laboratory on accessory replicative helicases is funded by the Biotechnology and Biological Sciences Research Council (grant number BB/K00168X/1).

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Review: Accessory Replicati

Received 19 August 2014; Received in revised form 29 September 2014; Accepted 6 October 2014 Available online 13 October 2014 Keywords: DNA replication; recombination; mutation; transcription; DNA repair Abbreviations used: ssDNA, single-stranded DNA; dsDNA, double-stranded DNA.

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