REVIEWARTICLE

DNA replication and homologous recombination factors:acting together to maintain genome stability

Antoine Aze & Jin Chuan Zhou & Alessandro Costa &

Vincenzo Costanzo

Received: 30 November 2012 /Revised: 27 March 2013 /Accepted: 27 March 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Genome duplication requires the coordinated ac-tion of multiple proteins to ensure a fast replication with highfidelity. These factors form a complex called the Replisome,which is assembled onto the DNA duplex to promote itsunwinding and to catalyze the polymerization of two newstrands. Key constituents of the Replisome are the Cdc45-Mcm2-7-GINS helicase and the And1-Claspin-Tipin-Tim1complex, which coordinate DNA unwinding with polymerasealpha-, delta-, and epsilon- dependent DNA polymerization.These factors encounter numerous obstacles, such as endoge-nous DNA lesions leading to template breakage and complexstructures arising from intrinsic features of specific DNAsequences. To overcome these roadblocks, homologous re-combination DNA repair factors, such as Rad51 and theMre11-Rad50-Nbs1 complex, are required to ensure completeand faithful replication. Consistent with this notion, many ofthe genes involved in this process result in lethal phenotypeswhen inactivated in organisms with complex and large ge-nomes. Here, we summarize the architectural and functionalproperties of the Replisome and propose a unified view ofDNA replication and repair processes.

Introduction

Genome duplication is a key event in the life cycle of allproliferating organisms and its careful control is essential topreserve the physical integrity of chromosomes (Arias andWalter 2007). The main player in this process is theReplisome, an assembly of macromolecular machines that serve

two essential functions: (1) coupling parental duplex–DNAunwinding with daughter strand synthesis (Macneill 2012)and (2) integrating DNA damage response signals to mod-ulate fork progression, pausing, and restart (Errico andCostanzo 2012).

Eukaryotic Replisome assembly occurs in multiple steps,which are timed in accordance with cell cycle cues. During theG1 phase, the origin recognition complex transiently associ-ates with the Cdc6 initiator to recruit a Cdt1•Mcm2-7heptamer to DNA replication start sites (“origins”) (Boos etal. 2012). The end result of this reaction is the formation of atopological link between duplex DNA and two copies of thehexameric Mcm2-7 helicase, which are found tethered viatheir N-terminal ends. In this configuration, origins are “li-censed” for activation; however, the unwinding function ofthe Mcm2-7 enzyme remains dormant (Remus et al. 2009).Upon entry into S phase, multiple factors are recruited toactivate the replication origins by either associating with, orchemically modifying, the Mcm2-7 helicase (reviewed inLabib 2010). The events that lead to the opening of duplexDNA are still poorly understood at a molecular level.According to the current consensus model, the two Mcmparticles are thought to move apart following DNA melting,to travel at the front of the Replisome (Botchan and Berger2010; Yardimci et al. 2010).

Helicase activation depends on the association of theReplisome component, Cdc45 (Tercero et al. 2000), withMcm2-7 and the concomitant recruitment of the GINS assem-bly (Gambus et al. 2006) (together forming the CMG) (Moyeret al. 2006). Multiple factors contribute to this event. Forexample, a phospho-protein assembly acts as a GINS•Cdc45chaperone (called Sld2•Sld3•Dpb11 in yeast) (Zegerman andDiffley 2007), while also promoting origin deposition of theleading strand polymerase Pol ε (Muramatsu et al. 2010).Another key player is Mcm10, which transiently associateswith the CMG to promote polymerase α/Primase origin

A. Aze : J. C. Zhou :A. Costa (*) :V. Costanzo (*)Clare Hall Laboratories, London Research Institute,South Mimms, Herts EN63LD, UKe-mail: Alessandro.Costa@cancer.org.uke-mail: Vincenzo.Costanzo@cancer.org.uk

ChromosomaDOI 10.1007/s00412-013-0411-3

association and possibly to aid in DNA opening (Kanke et al.2012; van Deursen et al. 2012).

Additional factors travel with the Replisome at the fork.One example is the replication pausing complex, composed ofTipin, Tim1, and And1 (Errico et al. 2009) and Claspin(Nedelcheva et al. 2005), structural proteins that tether theCMG helicase to the replicative polymerases and couple theiractivities (Fig. 1). These factors play a primary role inmaintaining chromosomal integrity under replication stressconditions, as they keep the CMG from translocating whenreplicative polymerases stall (Errico and Costanzo 2012).Other Replisome-associated factors are Topoisomerase IB(Gambus et al. 2006) that relieves the positive supercoilsaccumulating ahead of the replication fork (Vos et al. 2011)and the FACT histone chaperone complex (Gambus et al.2006), which has been implicated in parental nucleosomedisassembly ahead of the fork or in daughter strand nucleo-some reassembly at the back of the Replisome (Abe et al. 2011;Winkler and Luger 2011).

In this review, we aim at building an architectural frame-work to help describe the function of the Replisomeunperturbed or engaged in the interaction with the DNArepair machineries. We discuss the known structural fea-tures of the isolated Replisome components as well as theirassemblies, with a focus on the mechanisms for helicaseactivation and inactivation, and the roles of the Replisomepausing complex in modulating helicase/polymerasecrosstalk. We then describe the known relationships be-tween the Replisome and the DNA repair machinery fo-cusing on the role of DNA damage response proteins andhomologous recombination factors in unchallenged andperturbed DNA replication.

The building of a Replisome

Mcm2-7 activation requires large structural rearrangements

The engine of the replicative helicase is formed by six distinct,however, related polypeptides (Mcm2, 3, 4, 5, 6, 7)(Vijayraghavan and Schwacha 2012) that contain two do-mains: an N-terminal DNA interacting collar (Fletcher et al.2003) and a C-terminal motor domain that belongs to thesuperfamily of AAA+ ATPases (Brewster et al. 2008;Neuwald et al. 1999). These modules form two stacked ringsthat spool DNA through their aligned central cavity (Fletcheret al. 2003) with a 3’→5’ polarity (Chong et al. 2000; Kelmanet al. 1999) via a steric exclusion mechanism (Fu et al. 2011)and through an intricate allosteric network involving four poreloops per protomer (Barry et al. 2009). Indeed, limited DNAunwinding activity by the isolated Mcm2-7 complex has beenobserved in vitro, only in a small number of species and ina narrow window of buffer conditions (Bochman andSchwacha 2008), consistent with the idea that the recruit-ment of activators is required to stimulate the helicasefunction (Botchan and Berger 2010). In agreement with thisnotion, studies on recombinant Drosophila (Ilves et al. 2010;Moyer et al. 2006) or human proteins (Kang et al. 2012)indicate that both the ATPase and DNA unwinding functionsare greatly enhanced when the Mcm2-7 is co-expressed withGINS and Cdc45, whose association must induce activatingstructural rearrangements within the helicase subunits (Ilves etal. 2010). Three-dimensional electron microscopy explainsthe nature of this conformational transition. The isolatedMcm2-7 complex was shown to form an open lock-washerring containing a gap in between Mcm2 and Mcm5 (Costa etal. 2011; Lyubimov et al. 2012), also predicted in earlierbiochemical studies (Bochman and Schwacha 2008, 2010).In the context of the CMG holoenzyme instead, the Mcm2-7helicase is closed, with Cdc45 and GINS binding across theMcm2/5 gate, effectively working as a latch that compressesthe hexameric ring, sealing its gap (Costa et al. 2011).

Unique features of the Mcm2-7 ATPase assembly explainthe mechanistic implications of activator binding. Hexameric,ring-shaped helicases all contain bipartite active sites, withcatalytic residues contributed by neighbouring, closely packedsubunits, and usually need a set of six functional ATPases forunwinding (Lyubimov et al. 2011). TheMcm2-7 is peculiar inthat it tolerates inactivating changes in many of its subunits,while still working as a helicase; however, it requires a func-tional Mcm5/2 active site for unwinding (Bochman andSchwacha 2008, 2010; Ilves et al. 2010). So, as they forcethe motor ring into a closed configuration and lock the 2/5gate, GINS and Cdc45 turn on the unwinding function of theMcm2-7 helicase by acting to reconfigure the most criticalATPase active site in the hexamer, located at the 2/5 interface(Costa et al. 2011). This mechanism can be employed during

Mcm2-7AAA+AAA+

NTDNTD

Cdc45Cdc45

GINSGINS

Mrc1/Mrc1/ClaspinClaspin

Ctf4/And1Ctf4/And1Pol ε

CatalyticCatalyticNon-catalyticNon-catalytic

Pol α

PrimasePrimase

Tipin Tipin Tim1Tim1

CatalyticCatalytic

Non-catalyticNon-catalytic

Pοl δ

CatalyticCatalyticNon-catalyticNon-catalytic

?

??

Fig. 1 Interactions involving some key Replisome components. TheCtf4/And1-Tipin-Tim1-Mrc1/Claspin complex plays an important rolein bridging between the Cdc45-Mcm2-7-GINS helicase and the repli-cative polymerases alpha, delta, and epsilon

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origin activation but could also be used to modulate forkprogression when the Replisome encounters a DNA lesion(Hashimoto et al. 2011; Ilves et al. 2012).

In particular, the encounter of the helicase with a nick orgap in the template induces a dissociation of the GINSsubunit, leaving Cdc45 still bound to the Mcm2-7 helicase.This process might lead to a slowing down/halting of thehelicase progression in the presence of DNA damage. Themechanism by which this takes place is still unclear andmight involve the participation of the DNA damage check-point at local level (Hashimoto et al. 2011).

GINS, the central nexus in the eukaryotic replication fork

The GINS hetero-tetramer is composed of four gene prod-ucts (Sld5 and Psf1/2/3) evolved from one common archaealancestor through two sequential gene duplication events(Kamada 2012). The architecture of the GINS assembly re-flects its evolutionary history, as the four polypeptides form apseudo-twofold symmetric assembly, with an elongated struc-ture containing two small, coaxial apertures of unknown func-tion (Chang et al. 2007; Choi et al. 2007; Kamada et al. 2007).Accumulating evidence indicates that GINS not only works toactivate the Mcm2-7 helicase but also has a structural role inconnecting multiple key Replisome components (Gambus etal. 2009; Muramatsu et al. 2010).

Combined structural and genetic studies provide importantinsights into the architectural role of GINS. The preservationof three surface-exposed residue patches have been identifiedas vital for yeast survival, these include: (1) the Psf2 α helicaldomain, contacting the Mcm DNA interacting collar, (2) thePsf2 C-terminal beta domain interfacing with Cdc45, and (3)the water-accessible face of Sld5 (Choi et al. 2007), whichdoes not engage in any CMG contact but rather appears toproject from the CMG core, as the apex of a lateral protuber-ance, poised to interact with other Replisome factors (Costa etal. 2011). Indeed, GINS has been shown to associate withmultiple components involved in both leading and laggingstrand synthesis. For example, studies in yeast indicate thatGINS is recruited onto origins together with Pol ε (Muramatsuet al. 2010) by directly contacting the essential, non-catalyticsubunit B, a configuration that is likely kept in the context ofthe moving Replisome. A direct interaction between GINSand Pol α/Primase has been detected in vitro by surfaceplasmon resonance (De Falco et al. 2007), while Ctf4 (theyeast ortholog of the Replisome pausing factor, And1—alsosee below) has been implicated in bridging the CMG and Polα/Primase by binding the GINS subunits, Sld5 and Psf2(Gambus et al. 2009).

It is remarkable that GINS, a small ~100-kDa assembly,can interface with two distinct replicative polymerases, Pol αand ε (although whether these interactions are concomitant isunknown). Multiple lines of evidence support the notion of a

role for GINS as a polymerase-bridging factor. For example, abiochemical study on human proteins indicates that all threereplicative polymerases co-elute with GST-tagged GINS,although this interaction is weak and fails to survive aglycerol gradient sedimentation step (Bermudez et al. 2011).Further support for direct leading and lagging strand coordi-nation by GINS comes from the ancestral archaeal replicationsystem. For example, GINS purified from Sulfolobussolfataricus can be pulled down by recombinant Primase(Marinsek et al. 2006), while endogenous, HIS-taggedThermococcus kodakarensis GINS can be co-purified withthe replicative DNA polymerase Pol D (Li et al. 2010)(an enzyme in part related to the catalytic subunit of Pol ε, seebelow) (Johansson and Macneill 2010). Overall, biochemicaldata on the archaeal Replisome mirror the observations in theeukaryotic system, supporting the notion that GINS is a keyarchitectural factor that tethers the Mcm2-7 helicase to thereplicative polymerases.

Cdc45, a catalytically dead exonuclease

Despite its key role during DNA replication initiation, forkelongation, and pausing, progress in our understanding ofthe molecular function of Cdc45 has been slow. Somestructural insights have been derived from a small angleX-ray scattering study (Krastanova et al. 2012) on the iso-lated human protein and from the EM structure of the fullCMG complex (Costa et al. 2011). According to both stud-ies, Cdc45 contains a globular core with protruding arms. Inthe context of the CMG holoenzyme, these arms interfaceeither with the GINS assembly or the N-terminal domain ofMcm2 and 5, engaging in intimate contacts that keep Cdc45well anchored to the helicase.

Like GINS, the Cdc45 protomer plays multiple roles withinthe Replisome, which go beyond a structural function duringthe activation of the replicative helicase. For example, Cdc45has been implicated in directly interacting with a number ofreplication factors including Sld3 (Kamimura et al. 2001)during a key step in the cascade of events that lead to originactivation or Mrc1/Claspin (Lee et al. 2003) that coordinateshelicase and polymerase activities during fork elongation.

Interestingly, the N-terminal domain of Cdc45 is homolo-gous to the phosphodiesterase domain of the prokaryotic RecJfactor (Sanchez-Pulido and Ponting 2011), a single-strandedspecific 5’→3’ exonuclease (Makarova et al. 2012), which isinvolved in the RecF double-strand break (DSB) repair path-way in bacteria (Lovett and Clark 1984). Close inspection ofthe catalytic site sequence of Cdc45 highlights the presence ofconserved inactivating mutations that target key residues in-volved in catalytic metal coordination in RecJ (Krastanova etal. 2012; Makarova et al. 2012). In agreement with thisobservation, recent biochemical studies on recombinant hu-man Cdc45, reported single-stranded DNA binding but failed

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to detect any nuclease or duplex–DNA binding activity(Krastanova et al. 2012), suggesting that this factor mightemploy a defunct exonuclease domain as a scaffold for track-ing on one strand at the replication fork.

The Cdc45/RecJ homology provides important insightsinto the evolution of the eukaryotic DNA replication ma-chinery. Indeed, many archaea contain a RecJ homolog thatcan be purified in a complex with endogenous GINS(Marinsek et al. 2006). Although in some archaea, RecJcontains inactivating amino acid changes as in Cdc45(Makarova et al. 2012) (or lacks the phosphodiesterase activesite altogether) (Marinsek et al. 2006), other species contain acatalytically active enzyme (Li et al. 2011). These data suggestthat a prokaryotic DSB repair system was hijacked (indepen-dently, more than once) in archaea to become part of the DNAreplication machinery, a role maintained through evolution.Taken together with the dissociation of the GINS factorfollowing an encounter with a nick in the template, whichforms a DSB at replication forks, this finding suggest thatthe DSB repair machinery is intimately linked to thereplication process.

The role of the single-stranded DNA-binding function ofCdc45 during DNA replication remains unclear. Given themarginal localization of Cdc45 within the CMG helicase,offset of the Mcm2-7 DNA interacting channel (Costa et al.2011), it can be postulated that Cdc45 might be involved intracking on one of the tails of the moving replication fork.Alternatively, Cdc45 might act as a brake for the helicaseduring fork pausing or collapse. For example, detection of aDNA lesion could trigger the disassembly of the GINSactivator factor, removing the latch from Mcm and causingthe Mcm2-5 gate to open. This event would cause thedisruption of a topological link between the helicase andthe leading strand. To prevent release of the helicase from thestalled replication fork, Cdc45 could employ its dead exonu-clease scaffold to clamp onto the DNA, in a “parked” config-uration, waiting for Replisome re-assembly and ready for forkrestart (Fig. 2). Two studies support the notion of a helicasebreak role for Cdc45: a recent work on yeast proteins (Bruckand Kaplan 2013), showing helicase polymerase uncouplingfor a DNA-binding-deficient Cdc45, and the studies on col-lapsed forks in Xenopus egg extracts (Hashimoto et al. 2011).

Polymerase α/Primase

The leading and lagging strand polymerases, Pol ε and δ,elongate DNA polymers starting from RNA–DNA primerssynthetized by the Pol α–Primase complex. De novo synthesiscan only be catalyzed by the Primase that produces short ~7–12 nucleotide RNA segments subject to limited extension bythe DNA polymerase α (Frick and Richardson 2001). Thishighly coordinated process occurs in the context of a tetramericcomplex containing two Primase subunits (Pri1 and Pri2) and

two Pol α subunits (Pol 1 and Pol 2). A combination of X-raycrystallography and electron microscopy using yeast or theorthologous archaeal proteins provide a good architecturalview of this tetrameric assembly (also known as primosome)(Pellegrini 2012).

In particular, the archaeal Primase serves as a model forthe architecture of the eukaryotic enzyme. Here, the twosubunits, named PriS and PriL, form a curved complex,with PriS containing the active site and PriL located distallyand not directly involved in catalysis, but rather poised tocontrol the length of the nascent RNA primer (Augustin etal. 2001; Lao-Sirieix et al. 2005a, b).

Pol α contains a B family-type catalytic subunit 1connected to a regulatory subunit 2, also found in Pol δ andε (Johansson and Macneill 2010). The C-terminal domain ofPol1 interacts with Pol2 forming a stable heterodimeric com-plex (Klinge et al. 2009) while the very C-terminal tail of Pol1directly tethers the Primase dimer (Kilkenny et al. 2012).Contacts between the globular catalytic core domain,CTD-Pol1•Pol2 and the Pri1•2 appear to be tenuous, resultingin a highly flexible structure (Nunez-Ramirez et al. 2011),representing a major challenge to high-resolution structuralcharacterization.

And1/Ctf4

Ctf4 (And1 in higher eukaryotes) was initially isolated inSaccharomyces cerevisiae via a protein affinity screen as aDNA polymerase α interactor (Miles and Formosa 1992).Although non-essential for cell viability, deletion of Ctf4leads to defects in DNA replication with cells displayingabnormal morphology and a marked reduction in the rate ofDNA replication. Recent evidence indicates that Ctf4/And1might have a dual function, bridging between the helicase andpolymerases within the unperturbed Replisome, but also beingpart of the replication pausing complex, including Claspin,Tim1 and Tipin, the protein assembly responsible for modu-lating helicase/polymerase crosstalk during replication forkstalling, pausing, and restart (Errico and Costanzo 2012).

Studies in yeast indicate that Ctf4 bridges between thePrimase-associated DNA polymerase α and the replicativehelicase component GINS (Gambus et al. 2009; Tanaka etal. 2009). In particular, pull-down experiments performedon recombinant proteins indicate that the C-terminal portionof Ctf4 directly interfaces the GINS subunits Psf2 and Sld5while also interacting with the catalytic subunit of Pol α(Gambus et al. 2009).

The yeast Ctf4 interaction network appears recapitulatedin metazoan systems. For example in human cells, the Ctf4ortholog And1 is required for the association of GINS withthe Mcm2-7 helicase in human cells (Im et al. 2009) whilein Xenopus And1 is found to directly interact with Pol α andbind Tim/Tipin (Errico et al. 2009). In summary, Ctf4/And1

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appears to not only provide an important architectural linkbetween the replicative helicase and polymerases, but alsobridge the Replisome with the Replisome pausing complexthat controls Replisome pausing and restart (Errico et al.2007). The molecular mechanism of fork progression mod-ulation remains to be elucidated.

Polymerase ε

The leading strand polymerase, Pol ε, is a four-memberenzyme containing a large catalytic subunit (1), an essen-tial, non-catalytic subunit (2), and two non-essential sub-units (3 and 4), characterized by a histone fold motif (Hoggand Johansson 2012). The catalytic subunit forms a large,globular head domain followed by an extended, flexible tailcomposed of subunits 2, 3, and 4, as shown by cryo-EMstudies (Asturias et al. 2006).

Although the catalytic subunit belongs to the same Bfamily of polymerases (as do the two other eukaryotic repli-cative polymerases), this protomer is peculiar in that it con-tains a C-terminal zinc finger appendix homologous to thearchaeal DNA polymerase D, preceded by a tandem repeat oftwo whole polymerase domains (Tahirov et al. 2009).Whereas the N-terminal repeat of the catalytic subunit con-tains canonical DNA polymerization and editing functionsfound in other B-family polymerases, the C-terminal repeatbears inactivating mutations that make it a catalytically deadpolymerase module. Similar to Cdc45, it is unclear whetherthis defunct enzyme is employed in the context of the moving

fork, as a single-stranded DNA tracking element or whether itengages DNA in other contexts, for example during replica-tion initiation (Muramatsu et al. 2010) and/or fork pausing.Surprisingly, studies in yeast indicate that the C-terminal,catalytically dead half of Pol ε is the only domain essentialfor viability (although cells bearing a truncation of the cata-lytic domain grow slower) (Dua et al. 1998; Dua et al. 1999;Feng and D'Urso 2001; Kesti et al. 1999). This notion iscoherent with the idea that the role of the two tandem poly-merase repeats can be uncoupled.

Equally complex domain architecture can be found insubunit 2, which contains three recognizable modules.Remarkably, the structure of the N-terminal region resemblesthe lid of an AAA+ ATPase (Nuutinen et al. 2008). Thisobservation is particularly tantalizing, as Pol ε subunit 2 is aknown interactor of the GINS complex (Muramatsu et al.2010), which in turn works to modulate the opening/closureof the Mcm2-7 DNA gate (Costa et al. 2011). When inactive,Mcm2-7 exists in an open-end configuration, which exposesone AAA+ active site surface, a potential interactor for the Polε AAA+ lid-like domain (however, it remains to be testedwhether a direct contact between Mcm2-7 and Pol ε occurs).Following a central predicted oligosaccharide/oligonucleotidebinding fold, the C-terminal region of subunit 2 contains acalcineurin-like phosphodiesterase domain (yet another deadnuclease domain), a feature common to all eukaryotic repli-cative DNA polymerases (Johansson and Macneill 2010).

Altogether, the complex evolutionary history of the Pol εmulticomponent enzyme is mirrored by an intricate network

Mcm2-7 7

Cdc45Cdc45

GINSGINS

Pol ε

Pol εGINSGINS

Cdc45Cdc45Mcm2-7 7

Parental

LaggingLaggingstrandstrand

Leading g strand

Fig. 2 A speculative mechanism for helicase halting in response tofork collapse. When the Cdc45-Mcm2-7-GINS helicase encounters asingle-stranded DNA lesion, GINS and Pol ε disengage from theReplisome, while Cdc45 and Mcm2-7 remain bound to the replicationfork. GINS disengagement likely causes the opening of the Mcm2-7

DNA gate, which in turn could promote extrusion of the leading strandtemplate. The single-stranded DNA binding function of Cdc45 couldhelp maintain the helicase tethered to the collapsed replication fork.According to our model, Cdc45 likely catches the leading strandtemplate, released upon the Mcm2-7 ring opening

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of interactions with other replication factors. Two Pol εprotomers contain a catalytically dead DNA processingdomain, whose function to date is only partially understood,but most likely involves single-stranded DNA engagement(Tahirov et al. 2009).

Claspin/Mrc1

In similar fashion to Ctf4 on the lagging strand, the Claspinfactor (mediator of replication checkpoint, Mrc1, in yeast)has been shown to provide a physical link between the DNAhelicase and Pol ε on the leading strand (Lou et al. 2008).Indeed, domain mapping experiments have shown that boththe N-terminal and C-terminal regions of Mrc1 have a directrole in engaging Pol ε. Interestingly, the N-terminal domainofMrc1 becomes phosphorylated in response toDNA damage(Alcasabas et al. 2001; Osborn and Elledge 2003), leading todissociation of this domain from Pol ε while the C-terminalregion remains anchored to the polymerase (Lou et al. 2008).The functional consequence of this structural rearrangementremains to be elucidated. Like Ctf4, Mrc1-depleted cells areviable but lead to DNA damage accumulation (Liu et al. 2006)and show a greatly reduced fork progression rate possibly dueto the higher frequency of Replisome dissociation from chro-matin (Szyjka et al. 2005; Tourriere et al. 2005). Furtherstudies will likely elucidate how cells can survive in theabsence of architectural factors such as Claspin or Ctf4, whichlink the CMG helicase and the replicative polymerases.

DNA polymerase δ

Mammalian DNA polymerase δ was initially characterisedusing the in vitro SV40 DNA replication system where,together with Pol α/Primase, it mediates DNA synthesison both leading and lagging strands at the replication fork(Waga and Stillman 1994). Pol δ was later found to prefer-entially act on the lagging strand, as shown by mutation rateanalysis in yeast strains that carry an exonuclease-deficientPol δ and a wildtype Pol ε (Nick McElhinny et al. 2008;Pursell et al. 2007). Nonetheless, deletion of the catalyticdomain of Pol ε in yeast does not affect cell viability (Kestiet al. 1999), suggesting that Pol δ can be found structurallyassociated with both replication strands, in the absence of adedicated leading strand polymerase.

Together with the other two replicative DNA polymerases,Pol δ shares the evolutionarily conserved catalytic domain andthe accessory subunit B. Pol δ contains two additional, how-ever, non-ubiquitous (Gerik et al. 1998), members: the C andD subunits, evolutionarily unrelated to Pol ε’s subunits 3 and4 (Liu et al. 2000). While subunit D is not required for mitoticgrowth in fission yeast or DNA synthesis in vitro (Podust et al.2002; Reynolds et al. 1998), it contains an important fine-tuning function that balances the polymerizing activity and the

3´-5´ proofreading capacity (Meng et al. 2010; Meng et al.2009). Indeed, upon DNA damage, ATR signaling-induceddegradation of the D subunit leads to an enhanced 3´-5´exonuclease activity at the expense of the nucleotide extensionrate, which is lower compared to the four subunit polymerase.

The crystal structures of the human Pol δ subunit 2 incomplex with the N-terminal region of the C-subunit and thebudding yeast catalytic core are available (Baranovskiy et al.2008; Swan et al. 2009). The small D subunit remains the onlystructurally poorly characterized region of the polymerasealthough pull-down assays indicate that it binds to the cata-lytic subunit (Li et al. 2006). The conserved subunit 2 in Pol δacts as a central hub in bringing the catalytic and the regula-tory elements of the polymerase together into a completeholoenzyme. Interestingly, all four subunits of Pol δ interactwith the PCNA sliding clamp, although each via distinctbinding domains. For example, biochemical studies have re-vealed that the zinc-coordinating C-terminal region of thecatalytic subunit is important for PCNA binding (Netz et al.2012). Pol δ subunits B, C, and D have also been reported tocontain different PCNA-interacting motifs (Bruning andShamoo 2004; Li et al. 2006; Lu et al. 2002). The reason formultiple distinct interactions between Pol δ and PCNA isunclear although it may contribute to the processivity of thepolymerase during translocation along the DNA.

Emerging roles of DNA damage repair and responsefactors in unchallenged and perturbed replication

Protecting replication forks and promoting Replisomestability

DNA replication progression is frequently impaired by sec-ondary DNA structures, covalent adducts, and DNA lesions.Several systems ensure correct duplication of genomic DNAin prokaryotic and eukaryotic organisms. In organismswhere replication starts from a single origin, restartingmechanisms assist fork progression by exploiting the ho-mologous recombination DNA repair machinery (Errico andCostanzo 2012). In eukaryotes the mechanisms underlyingthe function of DNA repair and DNA damage responseproteins in DNA replication are less clear. Moreover, thelinks between DNA damage response and repair factorswith Replisome components and their contribution to themaintenance of genome stability DNA replication are large-ly unknown. This is an aspect of DNA replication of highereukaryotes, which has been difficult to address due to thefact that many DNA repair genes are essential in metazoancells. Experiments performed in yeast have greatly contrib-uted to understand the role of some of the DNA damageresponse genes at stalled and collapsed replication forks.Replisome components are maintained stably associated to

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DNA to ensure rapid replication resumption when replica-tion forks stall. To this end, numerous proteins, which arenot essential for DNA synthesis, are recruited to the repli-cation fork via interaction with members of the Replisome.Among these proteins, Timeless/Tim1/Tof1, Tipin/Csm3together with Claspin/Mrc1 have been identified, both inyeast and higher eukaryotes, as members of the replicationpausing complex that contributes both to fork stabilizationand to checkpoint activation. Tim1, Tipin, and Claspin areconsidered mediators of the ATR signalling cascade, beingimportant for the efficient phosphorylation of the effectorkinase Chk1. This pathway is essential to prevent the loss ofReplisome components from stalled forks.

The ATR-Chk1 pathway coordinates several mechanismsthat contribute to maintain replication fork stability. The ATR-Chk1 signalling cascade is required to maintain replicativepolymerases bound to forks to regulate branch migratinghelicases such as Blm and to fine tune homologous recombi-nation (HR), either positively or negatively (Cimprich andCortez 2008). The absence of a functional checkpoint leadsto Replisome dissociation and formation of aberrant DNAstructures that are processed by recombination proteins andexonucleases (Cotta-Ramusino et al. 2005; Segurado andDiffley 2008).

The Tipin/Tim1 complex also has a direct role in preserv-ing Replisome integrity by preventing excessive unwinding ofDNA at stalled replication forks. It is possible that the com-plex directly promotes the coupling between helicase andpolymerases. The interaction of these proteins with severalReplisome components, including polymerases and theCdc45–MCM–GINS (CMG) helicase complex indicates thatTipin, Tim1, and Claspin play a major structural role at stalledforks (Errico and Costanzo 2012). In contrast to yeast cells,the absence of Tipin/Tim1 is lethal in mammalian organismsand leads to the accumulation of chromosomal breakage evenin the absence of apparent DNA damage (Chou and Elledge2006). It is possible that Tipin/Tim1 assists continuous forkrestart by promoting Pol α re-priming on the leading strand(Errico et al. 2009). These events might be frequent due to theformation of endogenous DNA lesions even in the absence ofexogenous DNA damaging insults (Lindahl and Barnes2000). In line with the proposed role, recent evidence showedthat Tim1 is able to increase the activity of all major DNApolymerases and to decrease the activity of MCM helicase(Cho et al. 2013).

Homologous recombination factors and replication forkprogression

Genetic inactivation of many DNA recombination genes islethal at very early stages of development in higher eukary-otes. This suggests that DNA recombination genes are prob-ably required to assist DNA replication or to correct problems

encountered by the replication machinery more frequently inhigher eukaryotes than in simpler organisms. Although thefunction of many DNA repair factors following DNA damageis known the biochemical mechanisms underlying their func-tion during unchallenged DNA replication in vertebrate cellsare poorly understood. For example, inactivation of importanthomologous recombination genes such as Mre11, Rad50, orNbs1 is lethal in mice, indicating that these are required forcell survival in complex organisms (Errico and Costanzo2012). Moreover, replacement of Mre11 with an allele thatdoes not have nuclease activity causes phenotypes that areindistinguishable from those of mice null for Mre11 (Buis etal. 2008). In contrast, mutations in the nuclease domain ofMre11 in S. cerevisiae have a limited effect and Mre11 nullcell are mostly viable (D'Amours and Jackson 2002).Therefore, while yeast mutants in many of the key DNA repairgenes are viable, loss of the same proteins in higher eukary-otes results in cell or embryonic lethality. The reasons behindthis discrepancy are unclear. It is possible that other pathwaysin lower eukaryotes compensate for the absence of HR genesin unchallenged conditions.

An important role attributed to HR genes in DNA repli-cation is the restart of collapsed replication forks formedwhen Replisomes encounter an obstacle or a discontinuity inthe template leading to the formation of a broken end. Themechanism by which HR promotes the rebuilding of aReplisome has been largely characterized in yeast cells.This pathway is also known as break-induced replication(BIR), and it is defined as the restart of DNA replicationfrom a DSB (Llorente et al. 2008).

The mechanisms underlying BIR have been clarified in S.cerevisiae genomes in which an artificial DSB is induced amitotically arrested cell to initiate long stretches of newlysynthesized DNA. This system provides a unique model offork repair by HR (Lydeard et al. 2010). This system has beenuseful to establish that most of the HR genes are required forefficient fork restart through BIR. Extensive DNA synthesisassociated with BIR requires both the leading and laggingstrand polymerases and all the components of the replicativehelicase, whereas the replication factors necessary to assemblea pre-replication complex at a replication origin are not re-quired (Lydeard et al. 2010).

An important difference with normal DNA replicationforks is that the replication apparatus built through HRprocess is mutagenic. DNA–polymerases α and δ are re-quired for the initial steps of DNA synthesis, whereasDNA–polymerase ε becomes involved only later (Lydeardet al. 2007). Pol 32, the accessory subunit of polymerase δ,is essential for BIR but not to the progression of the normalreplication fork. The mutagenic Replisome that is formedduring BIR results in large frame shifts, leading to genomicinstability. Therefore, although BIR is important to rescuethe lethality arising from a DSB formed at collapsed forks, it

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compromises the genetic stability of the rescued cells (Deemet al. 2011; Hicks et al. 2010).

Intriguingly, recent investigations in fission yeast showthat HR can restart forks arrested by a replication forkbarrier independently of a DSB (Lambert et al. 2010;Mizuno et al. 2009).

This mechanism is also potentially mutagenic as shownby the observation that recombination-restarted forks have aconsiderably high propensity to execute a U-turn at smallinverted repeats contributing to the generation of grosschromosomal rearrangements (Mizuno et al. 2013).

BIR, which requires coordinated repair and replicationevents might have a central role in vertebrate cells. Thepresence of repetitive sequences might facilitate this typeof repair allowing homologous pairing of the broken arm ofthe replication fork with DNA segments downstream orupstream of the lesion (Costanzo et al. 2009). This type ofrepair might be essential for cell survival in the presence ofcollapsed forks at the expense of genome stability.

Although the involvement of HR has been clearlyestablished in genetic systems, the detailed biochemical anal-ysis of the role of HR factors in DNA replication is still lessclear. Experiments in the vertebrateXenopus laevis egg extractcell free system have been helpful to study the biochemistry ofDNA damage response and DNA repair factors in eukaryoticDNA replication, overcoming survival issues related to theinactivation of these factors. These cell free systems allowextensive biochemical analysis and can reproduce basic cellcycle events such as chromatin formation, nuclear assembly,and semi-conservative DNA replication. Egg extracts haveproven a powerful tool for the in vitro study of both DNAreplication and cell cycle progression (Costanzo et al. 2009;Costanzo and Gautier 2003, 2004).

Using specific antibodies to deplete specific proteins, ithas been shown that Mre11 is required during unchallengedDNA replication to prevent accumulation of DSBs during asingle round of DNA replication (Costanzo et al. 2001).

More recently, this system has been useful to dissect therole in DNA replication of another DNA repair gene in-volved in homologous recombination such as Rad51, whichis the eukaryotic ortholog of RecA in Escherichia coli, andplays a central role during meiosis as well as in DSB repair.Rad51 is not essential in S. cerevisiae, and yeast cellsdeficient for Rad51, Rad52, and Rad54 are viable underunchallenged conditions, whereas Rad51 depletion resultsin cellular lethality in vertebrates (San Filippo et al. 2008).This suggests that Rad51 plays indispensable roles not onlyin meiotic chromosomal recombination but also in normalcell cycle in higher organisms. Chicken DT40 cells arrest atG2 phase even without exogenous DNA damage upon con-ditional knockdown of Rad51, which leads to the accumula-tion of single-stranded DNA lesions activating the G2/Mcheckpoint (Su et al. 2008). However, it was not clear how

these single-stranded DNA (ssDNA) regions were formed.Using Xenopus egg extracts to examine the role of Rad51during DNA replication, it was found that Rad51 binds tochromatin during DNA replication and that its binding ispartially suppressed by inhibition of replication originassembly. This indicated that a fraction of Rad51 binding tochromatin takes place after replication forks have beenestablished and that in addition to its well-known role inDSB repair, Rad51 might be required for DNA replication(Hashimoto et al. 2010).

To gain insight into the function of Rad51 at replicationforks, electron microscopic analyses (EM) of genomic rep-lication intermediates (RIs), recovered after psoralen-crosslinking of nuclei replicated extracts, was performed.EM samples showed a high frequency of RIs in Rad51-depleted extracts showing at least one ssDNA gap behindthe replication fork and the presence of ssDNA regionsdirectly at the fork (Hashimoto et al. 2010). These datashowed that Rad51 is directly required at DNA replicationforks for uninterrupted and accurate replication of undamagedtemplates. Rad51 could have a protective role towards nascentDNA chains and the observed extended ssDNA stretches atthe fork could result from increased susceptibility toexonucleolytic degradation. This hypothesis was confirmedby evidence that nascent DNA strands are actually degradedby Mre11 nuclease in the absence of Rad51 leading to theformation of ssDNA gaps (Hashimoto et al. 2010). Mre11-dependent degradation of nascent DNA at stalled forks prob-ably reflects a physiological role of Mre11 nuclease at forks.One hypothesis is that Mre11-dependent cleavage of the 3´end of the nascent DNA is required to free the stalled poly-merase and promote replication fork restart downstream of thestalling site. This would explain the essential role of Mre11nuclease observed in mouse cells. It is possible that Rad51limits the extent of the resection, which progresses to patho-logical levels in its absence.

Regulating Rad51 and Mre11 function

How Rad51 and Mre11 are regulated on replication forks isstill poorly understood. As Rad51 is mainly in complex withBRCA2, it is likely that this large protein coordinates thedifferent roles of Rad51 in replication and DSB repair(Pellegrini and Venkitaraman 2004).

BRCA2 contains a number of repeats that are critical forbinding to Rad51 called the BRC repeat. There is also ahelical domain, which adopts an alpha helical structure,consisting of a four-helix cluster core (alpha 1, alpha 8,alpha 9, alpha 10) and two successive beta-hairpins. Thealpha 9 and alpha 10 helices pack with the BRCA2 OB1domain, which consists of a five-stranded beta-sheet thatcloses on itself. An intriguing region is the tower domain,which adopts a secondary structure consisting of a pair of

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long, antiparallel alpha-helices (the stem) that support athree-helix bundle at their end, called the 3HB domain,which is similar to the DNA binding domains of the bacte-rial site-specific recombinases. The Tower domain has animportant role in the tumour suppressor function of BRCA2,and is essential for BRCA2 binding to DNA. However, howthe different domains work together is poorly understood(Pellegrini and Venkitaraman 2004; Pellegrini et al. 2002).

As recently shown, BRCA2 loads Rad51 onto replicationforks to prevent the nuclease activity of Mre11 fromdegrading stalled replication forks (Schlacher et al. 2011).It is likely that BRCA2-dependent assembly of Rad51 ontostalled replication forks is required to prevent Mre11-mediated degradation of nascent DNA. BRCA2 role inDNA replication might be even more important for chromo-some integrity than its role in DSB repair. However, thereare some unresolved questions arising form these studies. Itis unclear what makes forks stall so frequently in the ab-sence of DNA damaging agents. The mechanism by whichBRCA2 loads Rad51 is also unclear. BRCA2 might loadRad51 onto regressed arms formed at reversed forks arisingin conditions that halt Replisome progression. These re-versed forks would have DSB-like ends available for resectionand Rad51 binding (Fig. 3a). This process might facilitateDNA damage bypass in the presence of fork stalling lesion(Fig. 3b). Alternatively, BRCA2might directly promote Rad51loading onto ssDNA gaps that might form frequently duringDNA replication (Jensen et al. 2010). This role for BRCA2might explain the wider requirement for HR-mediated

processes during unchallenged DNA replication. The clari-fication of these alternative models awaits further studies.

Responding to template breakage during DNA replication

Although we are beginning to understand how the Replisomesdeal with DNA lesions that halt the progression of replicationforks, we have limited knowledge of the molecular eventsoccurring during fork restart, especially in higher eukaryotes.The behavior of Replisome components and DNA repairfactors on unrepaired nicks at the passage of the forks hasbeen recently addressed at the biochemical level. In particular,the behavior of the CMG complex subunits Mcm2-7, Cdc45,and GINS was analyzed and it was found that the GINSsubunit and Pol ε are specifically lost upon induction ofssDNA lesions in the template (Hashimoto et al. 2011).Intriguingly, it was found that the Mcm2-7 helicase ismaintained on DNA and replication forks are then restoredin a Rad51 and Mre11-dependent fashion. In this process, theGINS and Pol ε are reloaded onto forks to restart replication.The uncoupling of GINS from the CMG complex was unex-pected, considering that Cdc45 and GINS are recruited ontoreplication forks interdependently during the initiation ofDNA replication. The release of GINS at the passage of thefork across a discontinuous template might be due to thestructural configuration that the GINS factor adopts withinthe CMG complex (Fig. 2).

Importantly, a consequence of GINS detachment wouldbe the slowing of helicase progression owing to the loss of a

ParentalDNA

??

Rad 51Rad 51

BRCA2BRCA2 Mre11or other

nucleases

Rad 51Rad 51

BRCA2BRCA2

Mre11

Mcm2-7 7

??

a

b

Fig. 3 a A speculativemechanism for Rad51/BRCA2function at replication forks.During DNA replication,Rad51 might be loaded inBRCA2 dependent fashion ontossDNA gaps that accumulatebehind replication forks and onregressed arms formed atreversed forks. Rad51 bound toDNA might prevent Mre11dependent degradation ofnascent DNA. b Fromreplication fork collapse torestart. Transition from achicken foot DNA structure to arestarted replication fork. Itremains unknown whether thisprocess requires nucleolyticactivity or rather damagebypass. Damaged DNA isindicated by a red triangle

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major activator of the complex. This would also limit theextent of ssDNA accumulation potentially arising fromDNA unwinding in the absence of DNA synthesis. Thereloading of GINS onto the Mcm2-7–Cdc45 complex dur-ing fork restart could then reactivate the stalled helicase.

In these studies, it was also found that inhibition of Mre11activity impairs replication fork restart and Replisome integ-rity after fork collapse. These findings suggest that Mre11functions are coordinated with DNA replication factors toensure efficient DNA replication under stressful conditions.Consistent with this, cells lacking Mre11 nuclease wereshown to be sensitive to replication fork-stalling agents, indi-cating that Mre11 is involved in the repair of these structures(Buis et al. 2008). Therefore, Mre11- and Rad51-dependentfork repair leading to reloading of the GINS onto the Mcm2-7–Cdc45 complex still engaged with the DNA could besufficient to restore a functional CMG helicase complex andpromote replication fork restart following template breakagein higher eukaryotes.

Conclusions

Recent progress indicates that theDNA recombination/breakagerepair machinery and the DNA replication apparatusmight be more intimately linked than anticipated. The evo-lutionary history of various eukaryotic Replisome compo-nents is coherent with this emerging theme. In fact, multiplesubunits have been identified both within the replicativehelicase and polymerases, which are catalytically dead en-zymes, derived from nucleases, originally involved for exam-ple in the prokaryotic double-strand break repair pathway.Likewise, multiple Replisome components, includingCtf4/And1 and Mrc1 have been in turn described as havingan architectural function within the unperturbed, movingReplisome, or playing a key role in modulating forkhalting/restart in the context of the Replisome pausing com-plex. Use of the same players during both elongation andDNA damage response suggests that perturbations toReplisome progression are more frequent than expected. Therequirement of homologous recombination factors duringDNA replication suggests that DNA templates are highlyvulnerable to breakage that might irreversibly halt replicationprogression. The frequent accumulation of disruptive DNAlesions might explain the hijack of the DSB repair factors bythe Replisome machinery. Alternatively, the evolutionary so-lutions found to replicate and process the DNA might havebeen subsequently adopted by the DNA damage repair ma-chinery to ensure maintenance of DNA integrity as shown bythe conserved domain used by both apparatus. In both cases,the two functions need to run in parallel and chromosomereplication cannot be completed in the absence of DNA repaireven in the absence of apparent stress. As these functions are

essential for cell duplication and survival, the study of theirintegrated functionwill increasingly rely on biochemical mod-el systems such as the X. laevis egg extract coupled to ad-vanced imaging tools to uncover the unknown links betweenthese processes. These studies, combined with thestructural/enzymatic characterization of in vitro reconstitutedprotein assemblies, will provide a framework to describe thelink between DNA replication and repair transactions at amolecular level. These studies will be crucial to understandhow DNA structure is maintained and propagated.

Acknowledgments The authors should like to thank Adelina Daviesfor the critical reading of the manuscript. This work was funded byCancer Research UK. V.C. is also supported by the European ResearchCouncil (ERC) start-up grant (206281), the Lister Institute of Preven-tive Medicine and the European Molecular Biology Organization(EMBO) Young Investigator Program (YIP).

References

Abe T, Sugimura K, Hosono Y, Takami Y, Akita M, Yoshimura A,Tada S, Nakayama T, Murofushi H, Okumura K, Takeda S,Horikoshi M, Seki M, Enomoto T (2011) The histone chaperonefacilitates chromatin transcription (FACT) protein maintains nor-mal replication fork rates. J Biol Chem 286:30504–30512

Alcasabas AA, Osborn AJ, Bachant J, Hu F, Werler PJ, Bousset K,Furuya K, Diffley JF, Carr AM, Elledge SJ (2001) Mrc1 trans-duces signals of DNA replication stress to activate Rad53. NatCell Biol 3:958–965

Arias EE, Walter JC (2007) Strength in numbers: preventingrereplication via multiple mechanisms in eukaryotic cells. GenesDev 21:497–518

Asturias FJ, Cheung IK, Sabouri N, Chilkova O, Wepplo D, Johansson E(2006) Structure of Saccharomyces cerevisiae DNA polymeraseepsilon by cryo-electron microscopy. Nat Struct Mol Biol 13:35–43

Augustin MA, Huber R, Kaiser JT (2001) Crystal structure of a DNA-dependent RNA polymerase (DNA primase). Nat Struct Biol8:57–61

Baranovskiy AG, Babayeva ND, Liston VG, Rogozin IB, Koonin EV,Pavlov YI, Vassylyev DG, Tahirov TH (2008) X-ray structure ofthe complex of regulatory subunits of human DNA polymerasedelta. Cell Cycle 7:3026–3036

Barry ER, Lovett JE, Costa A, Lea SM, Bell SD (2009) Intersubunitallosteric communication mediated by a conserved loop in theMCM helicase. Proc Natl Acad Sci U S A 106:1051–1056

Bermudez VP, Farina A, Raghavan V, Tappin I, Hurwitz J (2011)Studies on human DNA polymerase epsilon and GINS complexand their role in DNA replication. J Biol Chem 286:28963–28977

Bochman ML, Schwacha A (2008) The Mcm2-7 complex has in vitrohelicase activity. Mol Cell 31:287–293

Bochman ML, Schwacha A (2010) The Saccharomyces cerevisiaeMcm6/2 and Mcm5/3 ATPase active sites contribute to the func-tion of the putative Mcm2-7 'gate'. Nucleic Acids Res 38:6078–6088

Boos D, Frigola J, Diffley JF (2012) Activation of the replicative DNAhelicase:breaking up is hard to do. Curr Opin Cell Biol 24:423–430

Botchan M, Berger J (2010) DNA replication: making two forks fromone prereplication complex. Mol Cell 40:860–861

Brewster AS, Wang G, Yu X, Greenleaf WB, Carazo JM, Tjajadia M,Klein MG, Chen XS (2008) Crystal structure of a near-full-length

Chromosoma

archaeal MCM: functional insights for an AAA+hexamerichelicase. Proc Natl Acad Sci U S A 105:20191–20196

Bruck I, Kaplan DL (2013) Cdc45 Protein-single-stranded DNA inter-action is important for stalling the helicase during replicationstress. J Biol Chem 288:7550–7563

Bruning JB, Shamoo Y (2004) Structural and thermodynamic analysis ofhuman PCNA with peptides derived from DNA polymerase-deltap66 subunit and flap endonuclease-1. Structure 12:2209–2219

Buis J, Wu Y, Deng Y, Leddon J, Westfield G, Eckersdorff M,Sekiguchi JM, Chang S, Ferguson DO (2008) Mre11 nucleaseactivity has essential roles in DNA repair and genomic stabilitydistinct from ATM activation. Cell 135:85–96

Chang YP, Wang G, Bermudez V, Hurwitz J, Chen XS (2007) Crystalstructure of the GINS complex and functional insights into its rolein DNA replication. Proc Natl Acad Sci U S A 104:12685–12690

Cho WH, Kang YH, An YY, Tappin I, Hurwitz J, Lee JK (2013)Human Tim-Tipin complex affects the biochemical properties ofthe replicative DNA helicase and DNA polymerases. Proc NatlAcad Sci U S A 110:2523–2527

Choi JM, Lim HS, Kim JJ, Song OK, Cho Y (2007) Crystal structureof the human GINS complex. Genes Dev 21:1316–1321

Chong JP, Hayashi MK, SimonMN, Xu RM, Stillman B (2000) A double-hexamer archaeal minichromosome maintenance protein is an ATP-dependent DNA helicase. Proc Natl Acad Sci U S A 97:1530–1535

Chou DM, Elledge SJ (2006) Tipin and Timeless form a mutuallyprotective complex required for genotoxic stress resistance andcheckpoint function. Proc Natl Acad Sci U S A 103:18143–18147

Cimprich KA, Cortez D (2008) ATR: an essential regulator of genomeintegrity. Nat Rev Mol Cell Biol 9:616–627

Costa A, Ilves I, Tamberg N, Petojevic T, Nogales E, Botchan MR,Berger JM (2011) The structural basis for MCM2-7 helicaseactivation by GINS and Cdc45. Nat Struct Mol Biol 18:471–477

Costanzo V, Gautier J (2003) Single-strand DNA gaps trigger an ATR-and Cdc7-dependent checkpoint. Cell Cycle 2:17

Costanzo V, Gautier J (2004) Xenopus cell-free extracts to study DNAdamage checkpoints. Methods Mol Biol 241:255–267

Costanzo V, Robertson K, Bibikova M, Kim E, Grieco D, GottesmanM, Carroll D, Gautier J (2001) Mre11 protein complex preventsdouble-strand break accumulation during chromosomal DNAreplication. Mol Cell 8:137–147

Costanzo V, Chaudhuri J, Fung JC, Moran JV (2009) Dealing withdangerous accidents: DNA double-strand breaks take centre stage.Symposium on Genome Instability and DNA Repair. EMBO Rep10:837–842

Cotta-Ramusino C, Fachinetti D, Lucca C, Doksani Y, Lopes M, SogoJ, Foiani M (2005) Exo1 processes stalled replication forks andcounteracts fork reversal in checkpoint-defective cells. Mol Cell17:153–159

D'Amours D, Jackson SP (2002) The Mre11 complex: at the crossroadsof dna repair and checkpoint signalling. Nat Rev Mol Cell Biol3:317–327

De Falco M, Ferrari E, De Felice M, Rossi M, Hubscher U, Pisani FM(2007) The human GINS complex binds to and specifically stimu-lates humanDNA polymerase alpha-primase. EMBORep 8:99–103

Deem A, Keszthelyi A, Blackgrove T, Vayl A, Coffey B, Mathur R,Chabes A, Malkova A (2011) Break-induced replication is highlyinaccurate. PLoS Biology 9:e1000594

Dua R, Levy DL, Campbell JL (1998) Role of the putative zinc fingerdomain of Saccharomyces cerevisiae DNA polymerase epsilon inDNA replication and the S/M checkpoint pathway. J Biol Chem273:30046–30055

Dua R, Levy DL, Campbell JL (1999) Analysis of the essentialfunctions of the C-terminal protein/protein interaction domain ofSaccharomyces cerevisiae pol epsilon and its unexpected abilityto support growth in the absence of the DNA polymerase domain.J Biol Chem 274:22283–22288

Errico A, Costanzo V (2012) Mechanisms of replication fork protec-tion: a safeguard for genome stability. Crit Rev Biochem Mol Biol47:222–235

Errico A, Costanzo V, Hunt T (2007) Tipin is required for stalledreplication forks to resume DNA replication after removal ofaphidicolin in Xenopus egg extracts. Proc Natl Acad Sci U S A104:14929–14934

Errico A, Cosentino C, Rivera T, Losada A, Schwob E, Hunt T,Costanzo V (2009) Tipin/Tim1/And1 protein complex promotesPol alpha chromatin binding and sister chromatid cohesion.EMBO J 28:3681–3692

Feng W, D'Urso G (2001) Schizosaccharomyces pombe cells lackingthe amino-terminal catalytic domains of DNA polymerase epsilonare viable but require the DNA damage checkpoint control. MolCell Biol 21:4495–4504

Fletcher RJ, Bishop BE, Leon RP, Sclafani RA, Ogata CM, Chen XS(2003) The structure and function of MCM from archaeal M.thermoautotrophicum. Nat Struct Biol 10:160–167

Frick DN, Richardson CC (2001) DNA primases. Annu Rev Biochem70:39–80

Fu YV, Yardimci H, Long DT, Ho TV, Guainazzi A, Bermudez VP,Hurwitz J, van Oijen A, Scharer OD, Walter JC (2011) Selectivebypass of a lagging strand roadblock by the eukaryotic replicativeDNA helicase. Cell 146:931–941

Gambus A, Jones RC, Sanchez-Diaz A, Kanemaki M, van Deursen F,Edmondson RD, Labib K (2006) GINS maintains association ofCdc45 with MCM in replisome progression complexes at eukary-otic DNA replication forks. Nat Cell Biol 8:358–366

Gambus A, van Deursen F, Polychronopoulos D, Foltman M, JonesRC, Edmondson RD, Calzada A, Labib K (2009) A key role forCtf4 in coupling the MCM2-7 helicase to DNA polymerase alphawithin the eukaryotic replisome. EMBO J 28:2992–3004

Gerik KJ, Li X, Pautz A, Burgers PM (1998) Characterization of thetwo small subunits of Saccharomyces cerevisiae DNA polymer-ase delta. J Biol Chem 273:19747–19755

Hashimoto Y, Ray Chaudhuri A, Lopes M, Costanzo V (2010) Rad51protects nascent DNA from Mre11-dependent degradation and pro-motes continuous DNA synthesis. Nat StructMol Biol 17:1305–1311

Hashimoto Y, Puddu F, Costanzo V (2011) RAD51- and MRE11-dependent reassembly of uncoupled CMG helicase complex atcollapsed replication forks. Nat Struct Mol Biol 19:17–24

Hicks WM, Kim M, Haber JE (2010) Increased mutagenesis andunique mutation signature associated with mitotic gene conver-sion. Science 329:82–85

Hogg M, Johansson E (2012) DNA Polymerase epsilon. SubcellBiochem 62:237–257

Ilves I, Petojevic T, Pesavento JJ, Botchan MR (2010) Activation ofthe MCM2-7 helicase by association with Cdc45 and GINS pro-teins. Mol Cell 37:247–258

Ilves I, Tamberg N, Botchan MR (2012) Checkpoint kinase 2 (Chk2)inhibits the activity of the Cdc45/MCM2-7/GINS (CMG) replica-tive helicase complex. Proc Natl Acad Sci U S A 109:13163–13170

Im JS, Ki SH, Farina A, Jung DS, Hurwitz J, Lee JK (2009) Assemblyof the Cdc45-Mcm2-7-GINS complex in human cells requires theCtf4/And-1, RecQL4, and Mcm10 proteins. Proc Natl Acad Sci US A 106:15628–15632

Jensen RB, Carreira A, Kowalczykowski SC (2010) Purified humanBRCA2 stimulates RAD51-mediated recombination. Nature467:678–683

Johansson E, Macneill SA (2010) The eukaryotic replicative DNApolymerases take shape. Trends Biochem Sci 35:339–347

Kamada K (2012) The GINS Complex: structure and function. SubcellBiochem 62:135–156

Kamada K, Kubota Y, Arata T, Shindo Y, Hanaoka F (2007) Structureof the human GINS complex and its assembly and functionalinterface in replication initiation. Nat Struct Mol Biol 14:388–396

Chromosoma

Kamimura Y, Tak YS, Sugino A, Araki H (2001) Sld3, which interactswith Cdc45 (Sld4), functions for chromosomal DNA replicationin Saccharomyces cerevisiae. EMBO J 20:2097–2107

Kang YH, Galal WC, Farina A, Tappin I, Hurwitz J (2012) Propertiesof the human Cdc45/Mcm2-7/GINS helicase complex and itsaction with DNA polymerase epsilon in rolling circle DNA syn-thesis. Proc Natl Acad Sci U S A 109:6042–6047

Kanke M, Kodama Y, Takahashi TS, Nakagawa T, Masukata H (2012)Mcm10 plays an essential role in origin DNA unwinding afterloading of the CMG components. EMBO J 31:2182–2194

Kelman Z, Lee JK, Hurwitz J (1999) The single minichromosomemaintenance protein of Methanobacterium thermoautotrophicumDeltaH contains DNA helicase activity. Proc Natl Acad Sci U S A96:14783–14788

Kesti T, Flick K, Keranen S, Syvaoja JE, Wittenberg C (1999) DNApolymerase epsilon catalytic domains are dispensable for DNAreplication, DNA repair, and cell viability. Mol Cell 3:679–685

Kilkenny ML, De Piccoli G, Perera RL, Labib K, Pellegrini L (2012) Aconserved motif in the C-terminal tail of DNA polymerase alphatethers primase to the eukaryotic replisome. J Biol Chem287:23740–23747

Klinge S, Nunez-Ramirez R, Llorca O, Pellegrini L (2009) 3D archi-tecture of DNA Pol alpha reveals the functional core of multi-subunit replicative polymerases. EMBO J 28:1978–1987

Krastanova I, Sannino V, Amenitsch H, Gileadi O, Pisani FM, Onesti S(2012) Structural and functional insights into the DNA replicationfactor Cdc45 reveal an evolutionary relationship to the DHHfamily of phosphoesterases. J Biol Chem 287:4121–4128

Labib K (2010) How do Cdc7 and cyclin-dependent kinases trigger theinitiation of chromosome replication in eukaryotic cells? GenesDev 24:1208–1219

Lambert S, Mizuno K, Blaisonneau J, Martineau S, Chanet R, Freon K,Murray JM, Carr AM, Baldacci G (2010) Homologous recombi-nation restarts blocked replication forks at the expense of genomerearrangements by template exchange. Mol Cell 39:346–359

Lao-Sirieix SH, Nookala RK, Roversi P, Bell SD, Pellegrini L (2005a)Structure of the heterodimeric core primase. Nat Struct Mol Biol12:1137–1144

Lao-Sirieix SH, Pellegrini L, Bell SD (2005b) The promiscuousprimase. Trends Genet: TIG 21:568–572

Lee J, Kumagai A, Dunphy WG (2003) Claspin, a Chk1-regulatoryprotein, monitors DNA replication on chromatin independently ofRPA, ATR, and Rad17. Mol Cell 11:329–340

Li H, Xie B, Zhou Y, Rahmeh A, Trusa S, Zhang S, Gao Y, Lee EY, LeeMY (2006) Functional roles of p12, the fourth subunit of humanDNA polymerase delta. J Biol Chem 281:14748–14755

Li Z, Santangelo TJ, Cubonova L, Reeve JN, Kelman Z (2010)Affinity purification of an archaeal DNA replication protein net-work. mBio 1

Li Z, Pan M, Santangelo TJ, Chemnitz W, Yuan W, Edwards JL,Hurwitz J, Reeve JN, Kelman Z (2011) A novel DNA nucleaseis stimulated by association with the GINS complex. NucleicAcids Res 39:6114–6123

Lindahl T, Barnes DE (2000) Repair of endogenous DNA damage.Cold Spring Harb Symp Quant Biol 65:127–133

Liu L, Mo J, Rodriguez-Belmonte EM, Lee MY (2000) Identificationof a fourth subunit of mammalian DNA polymerase delta. J BiolChem 275:18739–18744

Liu S, Bekker-Jensen S, Mailand N, Lukas C, Bartek J, Lukas J (2006)Claspin operates downstream of TopBP1 to direct ATR signalingtowards Chk1 activation. Mol Cell Biol 26:6056–6064

Llorente B, Smith CE, Symington LS (2008) Break-induced replica-tion: what is it and what is it for? Cell Cycle 7:859–864

Lou H, Komata M, Katou Y, Guan Z, Reis CC, Budd M, Shirahige K,Campbell JL (2008) Mrc1 and DNA polymerase epsilon function

together in linking DNA replication and the S phase checkpoint.Mol Cell 32:106–117

Lovett ST, Clark AJ (1984) Genetic analysis of the recJ gene ofEscherichia coli K-12. J Bacteriol 157:190–196

Lu X, Tan CK, Zhou JQ, You M, Carastro LM, Downey KM, So AG(2002) Direct interaction of proliferating cell nuclear antigen withthe small subunit of DNA polymerase delta. J Biol Chem277:24340–24345

Lydeard JR, Jain S, Yamaguchi M, Haber JE (2007) Break-inducedreplication and telomerase-independent telomere maintenance re-quire Pol32. Nature 448:820–823

Lydeard JR, Lipkin-Moore Z, Sheu YJ, Stillman B, Burgers PM, HaberJE (2010) Break-induced replication requires all essential DNAreplication factors except those specific for pre-RC assembly.Genes Dev 24:1133–1144

Lyubimov AY, Strycharska M, Berger JM (2011) The nuts and bolts ofring-translocase structure and mechanism. Curr Opin Struct Biol21:240–248

Lyubimov AY, Costa A, Bleichert F, Botchan MR, Berger JM (2012)ATP-dependent conformational dynamics underlie the functionalasymmetry of the replicative helicase from a minimalist eukary-ote. Proc Natl Acad Sci U S A 109:11999–12004

Macneill S (2012) Composition and dynamics of the eukaryoticreplisome: a brief overview. Subcell Biochem 62:1–17

Makarova KS, Koonin EV, Kelman Z (2012) The CMG (CDC45/RecJ,MCM, GINS) complex is a conserved component of theDNA replication system in all archaea and eukaryotes. BiolDirect 7:7

Marinsek N, Barry ER, Makarova KS, Dionne I, Koonin EV, Bell SD(2006) GINS, a central nexus in the archaeal DNA replicationfork. EMBO Rep 7:539–545

Meng X, Zhou Y, Zhang S, Lee EY, Frick DN, Lee MY (2009) DNAdamage alters DNA polymerase delta to a form that exhibitsincreased discrimination against modified template bases andmismatched primers. Nucleic Acids Res 37:647–657

Meng X, Zhou Y, Lee EY, Lee MY, Frick DN (2010) The p12 subunitof human polymerase delta modulates the rate and fidelity ofDNA synthesis. Biochemistry 49:3545–3554

Miles J, Formosa T (1992) Evidence that POB1, a Saccharomycescerevisiae protein that binds to DNA polymerase alpha, acts inDNA metabolism in vivo. Mol Cell Biol 12:5724–5735

Mizuno K, Lambert S, Baldacci G, Murray JM, Carr AM (2009)Nearby inverted repeats fuse to generate acentric and dicentricpalindromic chromosomes by a replication template exchangemechanism. Genes Dev 23:2876–2886

Mizuno K, Miyabe I, Schalbetter SA, Carr AM, Murray JM (2013)Recombination-restarted replication makes inverted chromosomefusions at inverted repeats. Nature 493:246–249

Moyer SE, Lewis PW, Botchan MR (2006) Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryoticDNA replication fork helicase. Proc Natl Acad Sci U S A103:10236–10241

Muramatsu S, Hirai K, Tak YS, Kamimura Y, Araki H (2010) CDK-dependent complex formation between replication proteinsDpb11, Sld2, Pol (epsilon}, and GINS in budding yeast. GenesDev 24:602–612

Nedelcheva MN, Roguev A, Dolapchiev LB, Shevchenko A, TaskovHB, Shevchenko A, Stewart AF, Stoynov SS (2005) Uncouplingof unwinding from DNA synthesis implies regulation of MCMhelicase by Tof1/Mrc1/Csm3 checkpoint complex. J Mol Biol347:509–521

Netz DJ, Stith CM, Stumpfig M, Kopf G, Vogel D, Genau HM, StodolaJL, Lill R, Burgers PM, Pierik AJ (2012) Eukaryotic DNA poly-merases require an iron-sulfur cluster for the formation of activecomplexes. Nat Chem Biol 8:125–132

Chromosoma

Neuwald AF, Aravind L, Spouge JL, Koonin EV (1999) AAA+: A classof chaperone-like ATPases associated with the assembly, operation,and disassembly of protein complexes. Genome Res 9:27–43

Nick McElhinny SA, Gordenin DA, Stith CM, Burgers PM, KunkelTA (2008) Division of labor at the eukaryotic replication fork.Mol Cell 30:137–144

Nunez-Ramirez R, Klinge S, Sauguet L, Melero R, Recuero-ChecaMA, Kilkenny M, Perera RL, Garcia-Alvarez B, Hall RJ, NogalesE, Pellegrini L, Llorca O (2011) Flexible tethering of primase andDNA Pol alpha in the eukaryotic primosome. Nucleic Acids Res39:8187–8199

Nuutinen T, Tossavainen H, Fredriksson K, Pirila P, Permi P, PospiechH, Syvaoja JE (2008) The solution structure of the amino-terminaldomain of human DNA polymerase epsilon subunit B is homol-ogous to C-domains of AAA+ proteins. Nucleic Acids Res36:5102–5110

Osborn AJ, Elledge SJ (2003) Mrc1 is a replication fork componentwhose phosphorylation in response to DNA replication stressactivates Rad53. Genes Dev 17:1755–1767

Pellegrini L (2012) The Pol alpha-Primase complex. Subcell Biochem62:157–169

Pellegrini L, Venkitaraman A (2004) Emerging functions of BRCA2 inDNA recombination. Trends Biochem Sci 29:310–316

Pellegrini L, Yu DS, Lo T, Anand S, Lee M, Blundell TL, VenkitaramanAR (2002) Insights into DNA recombination from the structure of aRAD51-BRCA2 complex. Nature 420:287–293

Podust VN, Chang LS, Ott R, Dianov GL, Fanning E (2002)Reconstitution of human DNA polymerase delta using recombinantbaculoviruses: the p12 subunit potentiates DNA polymerizing ac-tivity of the four-subunit enzyme. J Biol Chem 277:3894–3901

Pursell ZF, Isoz I, Lundstrom EB, Johansson E, Kunkel TA (2007)Yeast DNA polymerase epsilon participates in leading-strandDNA replication. Science 317:127–130

Remus D, Beuron F, Tolun G, Griffith JD, Morris EP, Diffley JF (2009)Concerted loading of Mcm2-7 double hexamers around DNAduring DNA replication origin licensing. Cell 139:719–730

Reynolds N, Watt A, Fantes PA, MacNeill SA (1998) Cdm1, thesmallest subunit of DNA polymerase d in the fission yeastSchizosaccharomyces pombe, is non-essential for growth anddivision. Curr Genet 34:250–258

San Filippo J, Sung P, Klein H (2008) Mechanism of eukaryotichomologous recombination. Annu Rev Biochem 77:229–257

Sanchez-Pulido L, Ponting CP (2011) Cdc45: the missing RecJortholog in eukaryotes? Bioinformatics 27:1885–1888

Schlacher K, Christ N, Siaud N, Egashira A, Wu H, Jasin M (2011)Double-strand break repair-independent role for BRCA2 inblocking stalled replication fork degradation by MRE11. Cell145:529–542

Segurado M, Diffley JF (2008) Separate roles for the DNA damagecheckpoint protein kinases in stabilizing DNA replication forks.Genes Dev 22:1816–1827

Su X, Bernal JA, Venkitaraman AR (2008) Cell-cycle coordinationbetween DNA replication and recombination revealed by a verte-brate N-end rule degron-Rad51. Nat Struct Mol Biol 15:1049–1058

Swan MK, Johnson RE, Prakash L, Prakash S, Aggarwal AK (2009)Structural basis of high-fidelity DNA synthesis by yeast DNApolymerase delta. Nat Struct Mol Biol 16:979–986

Szyjka SJ, Viggiani CJ, Aparicio OM (2005) Mrc1 is required fornormal progression of replication forks throughout chromatin inS. cerevisiae. Mol Cell 19:691–697

Tahirov TH, Makarova KS, Rogozin IB, Pavlov YI, Koonin EV (2009)Evolution of DNA polymerases: an inactivated polymerase-exonuclease module in Pol epsilon and a chimeric origin ofeukaryotic polymerases from two classes of archaeal ancestors.Biol Direct 4:11

Tanaka H, Katou Y, Yagura M, Saitoh K, Itoh T, Araki H, Bando M,Shirahige K (2009) Ctf4 coordinates the progression of helicaseand DNA polymerase alpha. Genes Cells: Devoted Mol CellMech 14:807–820

Tercero JA, Labib K, Diffley JF (2000) DNA synthesis at individualreplication forks requires the essential initiation factor Cdc45p.EMBO J 19:2082–2093

Tourriere H, Versini G, Cordon-Preciado V, Alabert C, Pasero P (2005)Mrc1 and Tof1 promote replication fork progression and recoveryindependently of Rad53. Mol Cell 19:699–706

van Deursen F, Sengupta S, De Piccoli G, Sanchez-Diaz A, Labib K(2012) Mcm10 associates with the loaded DNA helicase at repli-cation origins and defines a novel step in its activation. EMBO J31:2195–2206

Vijayraghavan S, Schwacha A (2012) The eukaryotic mcm2-7 repli-cative helicase. Subcell Biochem 62:113–134

Vos SM, Tretter EM, Schmidt BH, Berger JM (2011) All tangled up:how cells direct, manage and exploit topoisomerase function. NatRev Mol Cell Biol 12:827–841

Waga S, Stillman B (1994) Anatomy of a DNA replication fork revealedby reconstitution of SV40 DNA replication in vitro. Nature369:207–212

Winkler DD, Luger K (2011) The histone chaperone FACT: structuralinsights and mechanisms for nucleosome reorganization. J BiolChem 286:18369–18374

Yardimci H, Loveland AB, Habuchi S, van Oijen AM, Walter JC(2010) Uncoupling of sister replisomes during eukaryotic DNAreplication. Mol Cell 40:834–840

Zegerman P, Diffley JF (2007) Phosphorylation of Sld2 and Sld3 bycyclin-dependent kinases promotes DNA replication in buddingyeast. Nature 445:281–285

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