Maintaining genome stability at the replication fork

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kinases CHK2 (also known as CHEK2) and CHK1 (also known as CHEK1), respectively, they act to amplify the checkpoint signal throughout the cell141,144.
REVIEWS

Maintaining genome stability at the replication fork Dana Branzei*and Marco Foiani*‡

Abstract | Aberrant DNA replication is a major source of the mutations and chromosome rearrangements that are associated with pathological disorders. When replication is compromised, DNA becomes more prone to breakage. Secondary structures, highly transcribed DNA sequences and damaged DNA stall replication forks, which then require checkpoint factors and specialized enzymatic activities for their stabilization and subsequent advance. These mechanisms ensure that the local DNA damage response, which enables replication fork progression and DNA repair in S phase, is coupled with cell cycle transitions. The mechanisms that operate in eukaryotic cells to promote replication fork integrity and coordinate replication with other aspects of chromosome maintenance are becoming clear. Autonomously replicating sequence A DNA element in the yeast genome that contains origins of replication.

Replication fork The branch point structure that forms during DNA replication between two template DNA strands, at which nascent DNA synthesis is ongoing.

*Fondazione IFOM, Istituto FIRC di Oncologia Molecolare, IFOM-IEO campus, Via Adamello 16, 20139 Milan, Italy. ‡ Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy. e-mails: [email protected]; marco.foiani@ ifom-ieo-campus.it doi:10.1038/nrm2852

Eukaryotic cells regulate the replication of their genomes in a highly complex manner that ensures the accurate and speedy duplication of the genetic information and preserves genome stability. DNA replication is tightly monitored to ensure that the genome is replicated just once per cell cycle, and that DNA replication is complete before mitosis begins. The sequence of events and many of the proteins involved in DNA replication are conserved throughout the eukaryotic world, although there are differences in the complexity of factors and in the mechanisms regulating DNA replication in different eukaryotes1,2. Replication initiates from multiple regions distributed along chromosomes (FIG. 1a). Certain organisms, such as the budding yeast Saccharomyces cerevisiae, have clear replicator sequences called autonomously replicating sequences (ARSs), whereas many other organisms have more relaxed DNA sequence requirements for the initiation events3. In S. cerevisiae, analysis of the replication time of all chromosomal segments suggests that origins differ in their time of initiation or ‘firing’, from early to mid and late S phase, as well as in their firing efficiency 4–6. Furthermore, the findings that there are more ARSs — which represent potential origins — than active origins on chromosomes, and that there is no phenotype associated with deleting replication origins, suggest that origins might be present in excess and used flexibly, with the passively replicated dormant origins being fired when major ones are inactivated1,6,7. All organisms studied so far seem to have an excess of origins3,6,8–10. The origins are marked by the formation of a pre-replicative complex (preRC) in G1, before DNA replication, through the binding of the origin recognition

complex (ORC) and the recruitment of additional replication factors, such as cell division control protein 6 (Cdc6), chromatin licensing and DNA replication factor 1 (Cdt1) and the minichromosome maintenance (MCM) helicase complex, which contains the six subunits Mcm2–Mcm7, to these sites. Because the preRC cannot be assembled later in the cell cycle, owing to the inhibitory activity of the S, G2 and M phase cyclin-dependent kinases (CDKs), the maximum number of origins available for an S phase is determined during the licensing state, which occurs in G1 when the preRC is formed. Furthermore, replication through an origin must remove or inhibit preRCs in order to prevent re-replication3,11. At each fired origin, two sister replication forks (RFs) are established that move away from the origin as the parental DNA duplex is unwound by the action of DNA helicases (FIG. 1b). The RFs can be hindered by physical impediments such as protein–DNA complexes and DNA damage. Different factors, such as the S. cerevisiae helicase Rrm3 (FIG. 1c)12 and proteins at the replication checkpoint13 (BOX 1), act to ensure the progression of RFs through natural fragile regions. When one RF is terminally blocked or arrested, firing of dormant or nearby origins ensures that replication is complete. DNA replication is an amazing undertaking as cells need to monitor the integrity of RFs while coupling replication with other cellular processes, such as chromatin reassembly, the inheritance of the epigenetic chromatin structure and the establishment of cohesion between sister chromatids. Cells replicating their DNA must also be able to initiate an adequate DNA damage response (DDR), execute DNA repair and replicate chromosome regions such as chromosome ends (telomeres) and

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preRC

preRC

MCM Origin of replication

Origin of replication

b MCM

MCM

Leading strand

MCM Discontinuous lagging strand

Fired origin Leading strand

c

Top2

RPA

LigI Polε Rad27 Mrc1 MCM RNase H Polα Dna2 Tof1 Csm3 Rrm3 RFC Polδ PCNA

Top1

Figure 1 | Replication initiation and progression. a | Replication begins from multiple Nature Reviews | Molecular Cell Biology origins, which are marked by the formation of a pre-replicative complex (preRC). b | Two replication forks (RFs), which are associated with the replisome that carries out DNA replication, are established at each fired origin. The minichromosome maintenance (MCM) helicase complex is shown ahead of the RFs, unwinding the duplex DNA. Replication is semidiscontinuous: DNA synthesis is continuous on the leading strand and discontinuous on the lagging strand, on which primers are elongated to form Okazaki fragments that are processed and ligated to one another. c | Numerous proteins are present at the RF. The MCM helicase unwinds the parental duplex, allowing access to the DNA polymerase-α (Polα) primase, replicative polymerase-δ (Polδ) and polymerase-ε (Polε) (which elongate the primers) and the replication processivity clamp proliferating cell nuclear antigen (PCNA; also known as Pol30), which is loaded by the clamp loader, the replication factor C (RFC) complex. Replication protein A (RPA) binds single-stranded DNA regions exposed at the RF or during lagging-strand synthesis. The discontinuous fragments synthesized on the lagging strand are processed by Rad27 (FEN1 in humans), Dna2 helicase, RNase H, Polδ and DNA ligase I (LigI). Several other factors associate with the RF in yeast and are represented: DNA topoisomerases 1 (Top1) and Top2, the checkpoint mediators mediator of replication checkpoint protein 1 (Mrc1), Top1-associated factor 1 (Tof1) and chromosome segregation in meiosis protein 3 (Csm3), and the Rrm3 helicase.

GINS complex An essential complex for DNA replication that promotes polymerase-ε loading and the activity of the MCM helicase.

ribosomal DNA containing repetitive DNA regions14–18. Several enzymatic activities in addition to the ones required for replication per se (FIG. 1c) are associated with the RF. These include checkpoint mediators that prevent the collapse of occasionally stalled RFs (BOX 1), nucleosome assembly and remodelling factors, DNA topoisomerases and factors required to establish sister chromatid cohesion. Genetic mutations that affect enzymes involved in replication result in an accumulation of DNA intermediates that trigger recombination and genome instability 19,20. Given the complexity of the tasks that must be completed during replication, and their need to be coordinated with one another, it is not surprising that defects

in DNA replication or its regulation underpin many human diseases and ageing. Therefore, understanding DNA replication and the pathways that suppress the instability of RFs is directly relevant to understanding the mechanisms by which cancers and other pathological disorders arise. here, we review the main causes of RF stalling and collapse, and the pathways that prevent RF instability. We begin with an overview of the basic aspects of RF progression and the topological mechanisms that assist DNA metabolism during DNA replication. We discuss the most important elements that affect RF integrity and how they can lead to a source of DNA breaks, and we explain the functions of checkpoints (BOX 1), DNA topoisomerases (BOX 2) and other specialized factors in maintaining RF stability and in preventing aberrant DNA transitions. Next, we consider the specialized pathways that deal with the distinct constraints imposed on DNA by different types of lesions and how they ensure RF progression. Finally, we comment on the consequences of treating genomic instability disorders with checkpoint or topoisomerase inhibitors, and on how the knowledge derived from genetic studies could reveal potential gene targets for disease prevention and therapy.

RF dynamics unwinding of the DNA is as essential a step for DNA replication initiation as it is for elongation. Formation of an active helicase at replication origins involves, besides preRC assembly, the recruitment of factors such as Cdc45 (which is essential for DNA replication initiation and travels with the RF) and the GINS complex, which interact with the core of the MCM replicative helicase complex, Mcm2–Mcm7 (ReFS 21,22). Origin firing also requires the activity of the S phase CDK and the Cdc7–Dbf4 kinase complex (also known as Dbf4-dependent kinase (DDK))23–26. The GINS complex is also important for the progression of RFs as it maintains the association between MCM and Cdc45 within the replisome27. The replisome is a multicomponent protein complex that is associated with the RF. It includes the MCM replicative helicase complex, replication elongation factors such as DNA polymerases (polymerase-α, polymerase-ε and polymerase-δ) and polymerase accessory factors (the clamp loader replication factor C (RFC) and the clamp proliferating cell nuclear antigen (PCNA; also known as Pol30 in S.cerevisiae)), mediator of replication checkpoint protein 1 (Mrc1) and the topoisomerase 1-associated factor 1 (Tof1)–chromosome segregation in meiosis protein 3 (Csm3) complex that is required for RF pausing (FIG. 1c). Since the chemistry of DNA synthesis dictates that the nucleotides are added to the 3′ end of the primer synthesized by the polymerase-α primase, the replication is semi-discontinuous: there is a leading strand, on which DNA nucleotides are added continuously to the initial primer, and a lagging strand, on which primers are synthesized and elongated throughout DNA synthesis to form Okazaki fragments. Once formed, Okazaki fragments are processed and ligated to the previous fragment (FIG. 1b).

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REVIEWS below), which are required to activate a robust DDR, the ubiquitylation pathway also responds to different types of defects that arise during DNA replication and contributes to the replication stress response (BOX 3).

Box 1 | The damage and replication checkpoint Replication stress or DNA damage Stop Stalled RF

ATR (or Mec1)

Terminal RF

DSB

ATM (or Tel1) and MRN (or MRX)

EXO1-mediated processing

EXO1-mediated processing

Checkpoints are cellular surveillance and signalling Nature pathways that detect DNACell lesions, Reviews | Molecular Biology such as single-stranded (ss)DNA or double-strand breaks (DSBs), and then boost a DNA damage response that ensures replication fork (RF) stabilization, DNA repair and cell cycle arrest. Ataxia telangiectasia mutated (ATM; telomere length regulation protein 1 (Tel1) in yeast) and ATM- and Rad3-related protein (ATR; mitosis entry checkpoint protein 1 (Mec1) in yeast) are key players in triggering the S phase checkpoint response. ATM (or Tel1) responds mostly to DSBs and is recruited to these through the MRN complex, which is composed of MRE11, RAD50 and NBS1 (or the MRX complex in yeast, which is composed of Mre11, Rad50 and Xrs2). ATM (or Tel1) and MRN (or MRX) prevent reversed RF formation and the formation of recombination structures derived from RFs that encounter DSBs (terminal RFs75; see the figure). ATR (or Mec1), however, responds mostly to stalled RFs with exposed ssDNA coated with replication protein A (RPA) to promote its stabilization (see the figure). In replication checkpoint mutants, the replisome dissociates from DNA29,30, leading to the formation of reversed RFs80 that are processed by exodeoxyribonuclease 1 (EXO1)82, which leads to long stretches of ssDNA73,74,83 (see the figure). The ssDNA–RPA that forms at stalled RFs acts to recruit two checkpoint complexes: ATR–ATR-interacting protein (ATRIP; Mec1–Ddc2 in yeast) and the checkpoint clamp loader RAD17 (Rad24 in yeast), which is required to load the proliferating cell nuclear antigen (PCNA)-like checkpoint clamp RAD9–RAD1–HUS1 (also known as 9-1-1; Rad17–Mec3–Ddc1 in yeast) onto ssDNA. 9-1-1 is then further phosphorylated by ATR (or Mec1) and this ensures robust checkpoint activation. Following ATM (or Tel1) and ATR (or Mec1) activation, mediator proteins are recruited to the site of DNA damage, and through phosphorylation of the effector checkpoint kinases CHK2 (also known as CHEK2) and CHK1 (also known as CHEK1), respectively, they act to amplify the checkpoint signal throughout the cell141,144. The functional orthologue of CHK1 in yeast is Rad53 but in yeast, following DNA damage, Mec1 activates both Rad53 and Chk1. The roles of Rad53 in stabilizing RFs are well documented29,30,73,74,83; Chk1 also appears to affect replication through damaged DNA145. The activation of the effector kinases triggers the checkpoint response through the phosphorylation of targets that are implicated in different, specific processes141,144.

Precatenane A cruciform junction that is formed by the intertwining of sister duplexes in the replicated portion of a replicon.

The progression of individual RFs can be stopped for various reasons, such as the presence of protein–DNA complexes or depletion of the nucleotide pool. In S. cerevisiae, proteins such as Mrc1 and Tof1 limit progression of the replisome under such circumstances28 and the checkpoint kinases mitosis entry checkpoint protein 1 (Mec1) and Rad53 (BOX 1) stabilize stalled RFs and prevent disassembly of the replisome29,30, thus preventing RF collapse. In addition to checkpoint proteins (BOX 1 and see

DNA topology unwinding of the duplex DNA induces differences in the topology and conformation of replicating DNA. The intertwining of the complementary strands in any DNA molecule is described by the linking number, which measures the number of times that one strand crosses the other strand in the DNA helix, and the number of times that one segment of double helix crosses another in higher-order superhelical structures31. The torsional stress created by the replication of any DNA segment cannot simply diffuse by the swivelling of the extremities of chromosomes because eukaryotic chromosomes are very large. Therefore, this tension can only be altered by DNA breakage and reunion reactions, which are mediated by specialized DNA nucleases called topoisomerases32 (BOX 2). Separation of the parental DNA strands by DNA helicases locally reduces the linking number of DNA molecules, generating compensatory positive torsional stress that can take the form of either supercoiling ahead of the RF or precatenanes that intertwine the two replicated duplexes behind the RF by swivelling the DNA at the RF branch point (FIG. 2a). Positive supercoils ahead of RFs can be removed in eukaryotes by type IB (TOP1) and type II (TOP2) topoisomerases (BOX 2), and this sustains the progression of the replication machinery (FIG. 2a). Indeed, both Top1 and Top2 were shown to travel with RFs in S. cerevisiae33. Precatenanes, however, do not affect helix unwinding and progression of the replication machinery. In fact, their formation diffuses the positive superhelical stress that accumulates in front of the RF. however, as precatenanes wind the daughter chromosomes around each other, they oppose partitioning of the chromosomes and therefore must be removed by topoisomerases. Catenanes and precatenanes can only be removed by the action of type II topoisomerases (BOX 2), which mediate the passage of an entire duplex through the other duplex that is present at the sites of sister chromatid juxtaposition32. Additional topological problems are generated when two RFs fuse at termination (FIG. 2b). The length of the unreplicated DNA becomes shorter as the RFs approach one another, such that the space that can contain the positive supercoils that are expected to result from RF movement becomes limited and RFs must rely on precatenane formation behind them in order to progress34 (FIG. 2b). The fusion of the two RFs will finally give rise to catenated sister chromatid junctions that have to be resolved before DNA segregation. limited Top1 and Top2 activity, and the increased requirements for DNA breakage–sealing reactions at catenated sister chromatid junctions, might explain the observation that late replicating regions are prone to breakage and exhibit fragility 13,35,36. Another situation that occurs during DNA synthesis is that RFs encounter transcription bubbles (FIG. 2c). Both head-on and in-line collisions between the replisome and the RNA polymerase (which is associated with the transcription bubble) have been shown to slow down

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f o c u S o n G E n o m E I n SR ta b I lE IW ty EV S Box 2 | DNA transitions mediated by DNA topoisomerases Topoisomerases are specialized enzymes that catalyze the passage of DNA single strands or double helices through one another (reviewed in ReFS 32,146). These transitions are mediated by transesterification reactions. The topoisomerase first becomes covalently attached to one of the ends of the DNA strand that has been attacked by the enzyme through a phosphotyrosine linkage. The hydroxyl group from one broken DNA end then attacks the phosphorus of the phosphotyrosine link, releasing the topoisomerase and rejoining the DNA strands. There are two general types of topoisomerases. Type I enzymes make single-strand breaks, allowing the uncut strand to pass through the break before resealing the nick. Type II enzymes are dimeric and coordinately introduce transient double-strand breaks in a DNA double helix, through which they then pass a segment of uncut DNA before resealing the break. Type II topoisomerases require ATP hydrolysis for their action, whereas type I topoisomerases do not. The energy released through ATP hydrolysis is used to promote topological transformations rather than to cleave or rejoin DNA. The two types of topoisomerase can be further divided into subfamilies (see the table): type IA (Top3), type IB (Top1), and type IIA (Top2). Type IA topoisomerases act to relax negatively supercoiled DNA and to pass one double helix through another if at least one of them contains a nick or a gap. The type IA topoisomerase, Top3, was proposed to act in concert with the RecQ helicases Sgs1 in yeast and Bloom syndrome protein (BLM) in humans to promote the resolution of double Holliday junctions91,110. Top1 enzymes are very efficient at relaxing both positively and negatively supercoiled DNA and probably provide the swivels for replication fork movement during replication by removing the positive supercoils in the unreplicated region. Top2 is required to decatenate chromosomes and precatenanes, and can relax both positive and negative supercoils, perhaps substituting for Top1 in its absence.

Topoisomerase subfamily

Representative eukaryotic member

Type IA

Yeast topoisomerase 3 (Top3) Mammalian topoisomerase 3α (TOP3A) and 3β (TOP3B)

Type IB

Yeast topoisomerase 1 (Top1) Mammalian topoisomerase 1 (TOP1) Mammalian mitochondrial topoisomerase 1

Type IIA

Yeast topoisomerase 2 (Top2) Mammalian topoisomerase 2α (TOP2A) and 2β (TOP2B)

Supercoil A contortion in DNA that is important for DNA packaging and DNA and RNA synthesis. Topoisomerases sense supercoiling and act to either generate or dissipate it by changing DNA topology.

Catenane An interlocked DNA molecule.

Late replication zone A DNA region that replicates late during S phase.

Triplex H‑DNA A DNA structure in which a DNA duplex associates with another DNA single strand, in either a parallel or antiparallel orientation.

Left‑handed Z‑DNA One of the three biologically active double helical structures of DNA. The others are A- and B-DNA.

the progression of RFs37,38. The observed pausing of RFs at transcription bubbles could be due to constraints in the mobility of the transcription machinery imposed by its association with the nuclear membrane39. In the case of a head-on encounter, the slowing down of RFs may also be caused by the excessive positive superhelicity generated in the downstream DNA by the movements of the two machineries37,40 (FIG. 2c). In addition to the expected predominant role of Top1 in resolving the positive supercoils41, a role for Top2 in preventing chromosome fragility at sites of S phase transcription has been recently described, whereby Top2 was proposed to mediate the formation of chromatin loops that insulate transcription units in S phase42.

Natural elements causing RF instability In addition to the DNA lesions occurring under physiological conditions (such as during hydrolysis or metabolism) or induced by external damaging agents43, the eukaryotic genome contains numerous natural impediments to replication, such as unusual DNA structures, late replication zones, DNA-binding proteins and transcription units. A common feature of these elements is that they induce the pausing of or completely block the progression of RFs, increasing the odds for RF breakage events. Several

examples of natural impediments to replication are given below, and their impact on RF stability and progression is discussed. Unusual DNA structures and chromosome fragility. DNA repeats, such as dinucleotide, trinucleotide, inverted, mirror and direct tandem repeats, can often undergo structural transitions that lead to the formation of alternative DNA structures, such as cruciforms, triplex H-DNA, left-handed Z-DNA and slipped-strand S-DNA, which could inhibit replication35. The continuation of DNA synthesis past these elements or the abnormal replication of the repeats, most often of trinucleotide repeats, has been proposed to lead to their expansion, and this phenomenon is responsible for many human diseases and hereditary disorders44–46. Expansion of repetitive elements has also been associated with chromosomal fragility — a cytogenetic term that describes DNA sequences that show gaps or breaks, following the partial inhibition of DNA synthesis47,48, that are often hotspots for chromosomal rearrangements47. Fragile sites are classified as common when they are present in all individuals and rare when they are present in less than 5% of the population. Rare fragile sites arise as a consequence of repeat expansion and have been associated with nearly 30 human hereditary disorders to date46. By contrast, common fragile sites do not have dinucleotide or trinucleotide repeats, are AT-rich and are normal components of chromosomes that are expressed on the inhibition of DNA replication47. The replication slow zones in yeast are also thought to represent common fragile sites13,36. Although there is no AT-rich bias in the replication slow zones, breakage at these sites is stimulated in the absence of an active ataxia telangiectasia and Rad3-related protein (ATR) checkpoint Mec1 in yeast), which is proposed to act by stabilizing the RFs that are prone to stall at these regions (see also below). Other replication-stress-sensitive loci have been reported in yeast and proposed to function analogously with common fragile sites. Notably, Ty elements and transfer RNA (tRNA) genes are a big source of genome rearrangements49,50. In yeast strains with reduced levels of polymerase-α, elevated levels of homologous recombination (hR)-mediated chromosome translocations frequently occur at certain Ty elements51. A chromosome region that contains multiple tRNA genes that are known to stall RFs is also prone to breakage and translocation events, particularly in replication checkpoint mutants52. Exactly what inhibits DNA replication at these fragile elements, leaving unreplicated or single-stranded (ss) DNA regions, is unknown. It might be the unusual conformations that these DNA regions are prone to adopt47. however, secondary structures should no longer be favourable as the RF approaches, owing to the positive superhelicity generated in front of the RF53 (FIG. 2a). hairpins or similar structures could form, however, on the lagging-strand template in the time window in which this becomes single stranded, thus interfering with the progression of the lagging-strand polymerase. Preferential instability of repeats on the lagging strand was indeed observed in studies performed on replicating plasmids containing palindromic fragments in both orientations

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REVIEWS Box 3 | Ubiquitin- and SUMO-mediated modifications in DNA replication Post-translational modification of proteins with ubiquitin or ubiquitin-like proteins, such as small ubiquitin-related modifier (SUMO), is evolutionarily conserved in all eukaryotes and affects many signalling networks, including the DNA damage response (DDR)147. The conjugation machinery involves an E1 activating enzyme, an E2 conjugating enzyme, an E3 ligase to enhance conjugation and/or mediate target specificity and proteases to deconjugate SUMO from target proteins. Recent studies provide evidence that ubiquitin-mediated processes contribute to the replication-stress response in different organisms. The F-box Saccharomyces cerevisiae protein Dia2 is a Skp1–cullin–F box (SCF) E3 ligase that is important for genome stability, travels with replication forks (RFs) and regulates RF progression under conditions of replication stress by interacting with and ubiquitylating replisome components (mediator of replication checkpoint protein 1 (Mrc1) and chromosome transmission fidelity protein 4 (Ctf4))148,149. Regulation of S phase by the ubiquitin proteasome system is also illustrated by the degradation of the DNA replication licensing factor Cdt1 (ReF. 150) and the minichromosome maintenance 2 (Mcm2)–Mcm7 (MCM) helicase complex22. Other examples include the degradation of the checkpoint kinase Chk1 after it is activated through phosphorylation by ataxia telangiectasia and Rad3-related protein (ATR; BOX 1)151,152, the control of the length of S phase by the E2 ubiquitin-conjugating enzyme UBCH7 (also known as UBE2L3) (ReF. 153) and the control of repair events by Fanconi anaemia and breast cancer type 1 susceptibility protein (BRCA1) E3 ligases154. The ubiquitylation and sumoylation of the polymerase clamp proliferating cell nuclear antigen (PCNA) at Lys164 has important roles in coordinating replication-associated repair events66,67,100,104–106,141, and different pathways of sumoylation promote RF integrity under conditions of DNA damage68,69. A new type of PCNA ubiquitylation occurs in both yeast and human cells in response to a deficiency in the DNA ligase I enzyme that is required to ligate Okazaki fragments formed during lagging-strand synthesis155, and the findings suggest that distinct ubiquitylation events on PCNA are required to trigger an efficient DDR caused by different defects in DNA replication.

Slipped‑strand S‑DNA A homoduplex DNA formed between two strands that have either the same number or a different number of repeats (usually triple repeats).

Replication slow zone A genetically encoded region that causes slower fork progression and also tends to accumulate convergent RFs and, thus, to represent the positions of preferential RF termination.

Ty element A eukaryotic transposable element that resembles retroviruses, with long terminal repeats at both ends in a direct orientation. The RNA intermediate formed by transcription of the Ty element is copied as DNA by a reverse transcriptase encoded by the Tyb gene of the Ty element. This DNA copy is then inserted into a new site in the yeast genome.

Transfer RNA A small RNA molecule that transfers a specific active amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation.

in Escherichia coli 54,55. Another inhibitory factor to replication could be the unusual chromatin structure around these DNA sequences. Indeed, DNA methylation and the subsequent heterochromatization of DNA regions around expanded repeats has been reported56. The binding of specific proteins to the structure-forming DNA sequence might also influence the stability of such elements. The S. cerevisiae hmo1 protein, a high mobility group protein implicated in chromatin architecture and organization57, binds to CAG repeat tracts to organize their chromatin structure and promote tract instability 58. Transcription and RF stability and dynamics. Occasional collisions between the replisome and the transcription machinery are inevitable. Two types of collision can occur: head-on, whereby the front edge of the RNA polymerase encounters the components of the RF on the lagging strand, and co-directional, whereby the components of the RF on the leading strand encounter the rear edge of the RNA polymerase (FIG. 2c). Both types of collision slow down the progression of RFs38. head-on collisions between the replisome and the RNA polymerase block replication, leading to RF demise and transcriptionassociated recombination, which may induce genome instability 19,59. By contrast, it has been suggested that the RNA transcript in in-line collisions can act as a primer to continue leading-strand synthesis60. This mechanism could be particularly relevant when leading strands encounter damaged DNA, as it would enable the lesion to be left behind the moving RF, thus precluding RF stalling and collapse (see below).

Replication blocking sites and termination. Other natural regions that slow down replication and cause temporal pausing or nearly complete replication arrest are RF barriers, at which RFs are purposefully stalled and replication arrested, and replication termination zones, at which slowing is topologically imposed by the two RFs approaching one another (FIG. 2b). RF barriers are often site-specific and they have been described at telomeres, centromeres, tRNA genes, rDNA arrays (which code for ribosomal RNAs) and the Schizosaccharomyces pombe mating type locus. A direct link between RF stalling and recombination-induced chromosomal rearrangements was shown when the RF barrier RTS1 was introduced into the S. pombe genome, ensuring efficient mating-type switching in the fission yeast by regulating the direction of replication61,62. Recent work has shown that preventing replication restart at RTS1, by stabilizing the stalled RF in a manner dependent on the PCNA-interacting protein Rtf2, allows completion of DNA synthesis by the converging RF63. In the absence of Rtf2, slow-moving RFs are established at RTS1 in a manner that is dependent on the helicase Srs2, which has been implicated in the regulation of recombination events during replication64,65. Small ubiquitin-related modifier (SuMO; Pmt3 in S. pombe; Smt3 in S. cerevisiae), the conjugation of which to S. cerevisiae PCNA is known to mediate the recruitment of Srs2 to sites of damage and favour certain recombination events there66–68, is also required for efficient termination at the RTS1 element 63. It is possible that having one of the RFs preferentially blocked by a RF barrier is a general mechanism of replication termination. Although studies of replication intermediates in S. cerevisiae did not reveal an important contribution of the sumoylation pathway in stabilizing stalled RFs at certain early origins of replication69, it is possible that such roles may be site-specific. Indeed, previous work in S. cerevisiae has shown that yeast with mutations affecting proteins in the sumoylation pathway, such as the SuMO-conjugating enzyme ubc9, the SuMO E3 ligase Mms21 (BOX 3) and the cohesin-like structural maintenance of chromosomes (SMC) complex, Smc5–Smc6, the sumoylation of which depends on Mms21 (ReF. 70), were not able to efficiently resolve the cruciform, hemicatenane-like intermediates that form during the replication of damaged templates69,71 (see below and FIG. 3). Since these structures resemble termination structures, formed when two replicons fuse, it has been proposed that sumoylation may be involved in the termination of replication69,71. The role of the replication checkpoint in promoting or sensing replication termination is also not clear. On one hand, under-replicated DNA, as well as fragility at replication slow zones and other common fragile sites, is increased in checkpoint-defective cells13,36. On the other hand, the under-replication occurring in Cdc14 mutations that are known to be defective in regulating transition through mitosis72 is not sensed by the replication checkpoint and cells progress to anaphase with unreplicated DNA18. The factors and the regulatory pathways promoting replication termination remain to be established.

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protein A (RPA), and they have a central role in promoting RF stabilization and DNA repair 29,30,73,74. By contrast, the ataxia telangiectasia mutated (ATM; telomere length regulation protein 1 (Tel1) in yeast) pathway responds mainly to double-strand breaks (DSBs) and is important in promoting the stability of RFs encountering DSB sites (terminal RFs)75 (BOX 1). When these checkpoint pathways are not functional, unusual structures accumulate owing to aberrant processing by the nuclease activities of the stalled, collapsed or terminal RFs73–75. Numerous studies also document the central role of the Mec1 and ATR pathways in preventing the expression of fragile sites13,36,52,76–79. When S. cerevisiae mutations for the checkpoint proteins Mec1 or Rad53 (BOX 1) are exposed to replication stress, the replisome dissociates from stalled RFs29,30. This is thought to be the initial step that then allows the nascent DNA chains to engage in pathological transitions, such as the formation of reversed RFs 80. It has been shown that RF reversal leads to topological constraints that prevent their further regression81 and perhaps generates additional substrates for topoisomerases. Once reversed, the RFs can be processed to generate the formation of DSBs, which could induce recombination or long stretches of ssDNA regions73,74,82,83 (BOX 1). The initial role in promoting RF stability starts with the RF pausing process itself, which is probably modulated by Mrc1 and Tof1 — two replisome components28 that also function as checkpoint adaptors and influence the activation of the checkpoint response following replication stress84–87 (BOX 1). Mrc1 and Tof1 seem to play an active role in promoting the pausing or stalling of RFs as, in their absence, the replisome progresses faster than it is able to synthesize DNA28. Mutations in Mrc1 and Tof1 also increase the fragility associated with expanded triple repeats79,88. Following the pausing of RFs, it is possible that the signal is transmitted to Mec1 and Rad53, which prevent replisome disassembly from stalled RFs. The replication checkpoint is also involved in regulating subsequent steps related to RF restart processes89–92 (see below).

Top1 Top2

Advancing RF

Positive supercoil

Top1 Top2 Top2 Positive supercoil

Precatenane

b Converging RFs

Positive supercoil Top1

Top2 Top2

Top2

Precatenane

Precatenane Top1 Top2

c

Positive supercoil Advancing RF

Transcription unit

Figure 2 | Topological transitions at the replication fork. a | Unwinding of the duplex DNA generates a positive supercoil ahead of the replication fork (RF), which is removed Reviews | Molecular CellRF, Biology mainly by topoisomerase 1 (Top1), but also by Top2Nature or precatenanes behind the the decatenation of which requires Top2. b | Positive supercoils must be converted into precatenanes at replication termination. As the RFs approach one another before fusion, the region available to contain positive supercoils diminishes and precatenanes form. c | The head-on encounter of a RF with a transcription unit leads to RF demise. The predominant topoisomerase involved in each situation is shown in red and the other in yellow.

Homologous recombination A type of genetic recombination in which DNA sequences are exchanged between two similar or identical strands of DNA.

The RF and the replication checkpoint The replication checkpoint stands out as the prime model of a regulator of replication that affects RF stability. Intertwined networks of sensors and transducers act to detect, transmit and amplify the damage or replicationstress signal, which leads to checkpoint activation (BOX 1). The ATR (Mec1 in yeast) pathway is activated by stalled RFs with exposed ssDNA regions coated with replication

Types of DNA damage that hinder the RF Different types of structural and chemical alterations in DNA can hinder the progression of RFs, leading to RF demise and the formation of DSBs that could trigger chromosomal aberrations. The distinct structural constraints imposed by the different lesions elicit the action of diverse damage tolerance pathways that restore RF progression or post-replicative repair, as discussed below. Most of these lesions also trigger the activation of the damage checkpoint. The roles of the checkpoint pathway in promoting the repair or damage tolerance events are complex and not fully elucidated87. The pathways operating during S phase to promote the repair or damage bypass of different types of lesions are discussed below. Bulky DNA lesions and damage tolerance pathways. Exogenous DNA damage, such as by ultraviolet (uV) irradiation and alkylating agents, and endogenous processes, such as hydrolysis, which leads to spontaneous DNA depurination, or reactive oxygen species that

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Lesion Repetitive element

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Amplification of the repeat

Figure 3 | Translesion synthesis- and template switch-mediated damage bypass Nature Reviews | Molecular Cell Biology mechanisms. Replication of damaged templates generates gaps on both strands. A replication fork (RF) with gaps formed on the leading strand owing to repriming events is shown in the rectangle, in which the structure on the left shows the traditional representation of the RF with a positive, upstream supercoil and the structure on the right shows the RF in a precatenane conformation. Damage bypass uses translesion synthesis (TLS) polymerases, which directly bypass the damage and can often induce DNA mutations, or a recombination-mediated mechanism called a template switch, which usually involves the newly synthesized sister chromatid or other sequences with homology to the single-stranded DNA region present in the gap. Template switching occurs through a series of events that, in principle, lead to the error-free bypass of lesions, as indicated in the box on the right. When the region to be copied contains repetitive elements, slippage and misalignment can lead to repeat contraction or amplification. An amplification event is highlighted in the box on the left. Top3, topoisomerase 3.

Heterochromatization The formation of a tightly packed form of DNA, which makes the DNA less accessible to protein factors that usually bind. Certain DNA elements, such as centromeres and telomeres, are heterochromatic.

Bulky lesion A DNA lesion in which the nucleotides carry bulky groups. Methylated DNA and thymine dimers caused by UV irradiation are examples.

induce base oxidation and DNA breaks, can lead to the formation of bulky lesions (for example, DNA adducts) that stall RFs93. In the semidiscontinuous model of replication (in which the leading strand is extended in a continuous manner but the lagging strand contains discontinuities (FIG. 1b)), blocking the leading replisome dictates that RF restart occurs at the original site of pausing. This can be achieved either by using specialized translesion synthesis (TlS) polymerases that can temporarily replace the replicative polymerase or by switching templates to the newly synthesized sister chromatid, which is made available by RF regression mechanisms or by hR-mediated strand invasion in the homologous duplex 94. hR-mediated

strand invasion involves the assembly and polymerization of Rad51 on the ssDNA that is exposed in proximity to the lesion. This leads to the formation of the Rad51 presynaptic filament , which can engage in homology search and invade the homologous region in the duplex to form a DNA joint, called a displacement loop (D-loop), which can then be extended through DNA synthesis (FIG. 3). These two mechanisms — TlS and template switch — constitute the two main pathways of damage tolerance, although whether they occur at or behind the RF is still a matter of debate. Recent work from several laboratories supports the conclusion that, in the presence of DNA damage, DNA synthesis is discontinuous on both strands60,92,95,96. The leading strand can restart using a primer that the primase makes on the leading strand, as well as on the lagging strand95. Alternatively, the mRNA transcript can be used as a primer by the leading-strand polymerase when it encounters the RNA polymerase of a transcription unit during inline collisions60,97. These mechanisms will leave the bulky lesion behind the RF, concealed in a gap, both on the leading and lagging strands. Gaps can later be filled in by TlS polymerases or template switch mechanisms, as mentioned above (FIG. 3). In this view, these damage tolerance pathways take place behind the RF. The damage replication checkpoint influences both branches of damage tolerance. The RAD9–RAD1–huS1 (also known as 9-1-1) damage checkpoint facilitates the recruitment of the TlS polymerase Polζ in S. cerevisiae and DinB (also known as Mug40) in S. pombe to sites of damage89,90, and damage-induced mutagenesis is partially dependent on the damage checkpoint 98–100. S. cerevisiae Rad53 and phosphorylation of the 9-1-1 complex on Rad9 by the ATM- and ATR-related Rad3 checkpoint kinase in S. pombe100 also promote error-free-mediated gap filling 91,100. Genetic studies conducted in yeast have already established that error-free-mediated damage bypass by template switching occurs by two main pathways: one mediated by Rad18–Rad5–Mms2–ubc13, referred to as error-free post-replication repair (PRR), and the other one mediated by Rad51–Rad52 (ReFS 101–103). These pathways cooperate to promote the bypass of the lesion through sister chromatid pairing, which leads to cruciform structures that resemble hemicatenanes68. Monoubiquitylation and polyubiquitylation of PCNA at the highly conserved residue lys164 (ReF. 104) differentially affects the two pathways of damage tolerance. Rad6and Rad18-dependent monoubiquitylation promotes TlS in both yeast and mammalian cells105,106, whereas ubc13– Mms2 and Rad5-dependent PCNA polyubiquitylation in yeast through lys63-linked chains is required for error-free PRR104,107,108 and template switch-mediated sister chromatid junction formation68. In yeast, PCNA is also sumoylated104 and biochemically these modifications occur independently of each other 66,67,104. however, in the absence of PCNA sumoylation, the ability of the error-free PRR pathway to promote template switching through sister chromatid junctions is impaired68, suggesting that PCNA sumoylation favours the damage bypass of lesions by enabling the use of factors belonging

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DNA adduct A piece of DNA that is covalently bonded to a chemical.

Homologous duplex A DNA duplex that shows homology with another DNA region or sequence and usually contains a DSB.

Presynaptic filament A nucleoprotein filament consisting of Rad51 molecules bound to ssDNA.

Hemicatenane A cruciform junction of two dsDNA molecules, in which one of the strands of one duplex passes between the two strands of the other duplex, and vice versa.

Microhomology Refers to homologous sequences that are extremely small, usually just a few base pairs long.

to the error-free branch of PRR. This interpretation is in agreement with genetic data in budding and fission yeasts, in which a lack of PCNA sumoylation was shown to suppress the damage sensitivity of error-free PRR mutants66,67,100. The exact mechanism underlying this phenomenon is unknown but, besides influencing the interacting partners of PCNA, sumoylation might affect the chromatin structure or the accessibility of factors such as Rad18 to sites of DNA lesions. In support of this, a recent study has found that the chromatin remodelling factor inositol-requiring protein 80 (Ino80) is important for efficient PCNA ubiquitylation following methyl methanesulphonate treatment, the recruitment of Rad18 and Rad51 to damaged RFs and the formation of sister chromatid junctions during replication of damaged templates109. The sister chromatid junctions formed through template-switch processes are resolved by the action of the RecQ helicases Sgs1 (in yeast) and Bloom syndrome protein (BlM; in humans), together with the topoisomerase Top391,110,111 (FIG. 3). Sumoylation mediated by ubc9 and the SuMO E3 ligase Mms21, and the Mms21associated SMC complex, was shown to counteract the accumulation of these structures69,71,112. understanding the factors contributing to the timely resolution of these structures is important as, in the absence of Sgs1 (or BlM), Top3 and other regulatory activities, the cruciform structures may become targets for endonucleases, leading to DSB formation and excessive recombination (known as hyper-recombination). As precatenane formation is coordinated with replication it is possible that, together with cohesion, precatenanes facilitate the interaction between sister chromatids and, thus, subsequently also facilitate template switching (FIG. 3). Template switching is mostly an error-free mechanism, but the replication of DNA fragments containing repetitive elements often leads to misalignments and the production of repeat rearrangements113 (FIG. 3). Indeed, factors implicated in promoting template switching, such as Rad6, Rad5 and Sgs1 (ReF. 68), were also shown to increase the instability of repeated sequences88,114. RFs containing hairpins or other secondary DNA structures can be preferentially contained in chromatin loops or in higher-order DNA conformations. Pairing of the initially blocked nascent chain with the sister chromatid (FIG. 3), or with a strand from a distant RF that contains microhomology elements and is brought nearby by chromatin looping events, could lead to genome rearrangements115,116. Recent studies in budding and fission yeast have shown that RF arrest in repeated sequences of nearby inverted repeats can lead to a switch of templates that generate dicentric chromosome intermediates, which, on segregation, lead to breakage and further chromosome rearrangements117,118. understanding how genome architecture affects replication-associated repair events is a fascinating topic in chromosome and cellular biology. Interstrand cross links and their repair. Covalent links between complementary DNA strands completely block RF progression by precluding strand separation. Interstrand cross links (ICls) are widely generated by

cancer chemotherapy and are also formed by cellular metabolites119. In mammalian cells, the repair of ICls involves the interplay of the Fanconi anaemia pathway, hR and TlS polymerases120. Cells from individuals afflicted with Fanconi anaemia, a rare disorder characterized by congenital abnormalities and an increased incidence of cancer, exhibit chromosomal fragility and are notoriously hypersensitive to ICl agents121. Indeed, the Fanconi anaemia pathway prevents the formation of replication-associated DSBs122. Fanconi anaemia proteins can be classified into three groups. Group I includes eight Fanconi anaemia proteins that constitute the core complex. This complex has ubiquitin ligase activity 121,123 and ubiquitylates FANCI and FANCD2, which constitute the group II proteins124 (FIG. 4). In addition, Fanconi anaemia-associated protein of 24 kDa (FAAP24), a protein that shares homology with the XPF family of endonucleases, interacts with the FANCM helicase of the Fanconi anaemia core complex, and is required for full levels of FANCD2 ubiquitylation in S phase or after damage125. ubiquitylation of FANCI–FANCD2 is essential for its ability to form foci on damaged chromatin, where other recombination proteins, such as RAD51, breast cancer type 1 susceptibility protein (BRCA1), and BRCA2 (also known as FANCD1), are recruited124. BRCA2, together with its interacting protein FANCN (also known as PAlB2) and the BRCA1-associated DNA helicase FANCJ (also known as BRIP1)126 are group III Fanconi anaemia proteins. These proteins do not affect FANCD2 ubiquitylation, but contribute to hR repair 127,128 (FIG. 4). The exact role of Fanconi anaemia proteins in repair is not yet understood, but genetic evidence suggests that they might act to create an appropriate substrate for hR or TlS129. This substrate may be reversed RFs, as FANCM was reported to have RF remodelling activity that can convert a RF into a four-way junction130–132. In S. cerevisiae, the helicase activity of the FANCM orthologue mutator phenotype protein 1 (Mph1) was shown to promote the accumulation of recombination intermediates that require the Smc5–Smc6 complex for resolution133. Recent studies using a cell-free system based on Xenopus laevis egg extracts analysed the replication of a plasmid containing a site-specific ICl134 (FIG. 4). In this system, the two RFs converge at the cross link, with both leading strands initially stalling 20–40 nucleotides away from the lesion, perhaps owing to topological constrains. One leading strand is then extended to within one nucleotide of the ICl, and dual incisions are made on both sides of the ICl on the parental DNA leading strand that contains the RF that stalled 20–40 nucleotides away from the lesion. Repair then occurs through TlS-mediated bypass of the lesion on the strand on which further elongation took place and by hR on the other strand134 (FIG. 4). ubiquitylated FANCI and FANCD2 were shown to be required for the nucleolytic incision near the ICl and for the TlS step135. The two RFs converging at the ICl site resemble the situation that occurrs at replication termination. Whether RF reversal is actively prevented by uncoupling the leading and the lagging strands of the converging

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b

S phase damage ATR ATRIP

ICL Replisome

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FANCA FANCB FANCC FAAP24 FANCE FANCF FANCG FANCM

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FANCI FANCD2

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FANCA FANCB FANCC FAAP24 FANCL FANCE FANCF FANCG FANCM FANCI FANCD2 Ub

Translesion synthesis Resection

RAD51 RAD52 RAD54 FANCJ BRCA1 FANCN BRCA2

Nucleotide excision repair Homologous recombination

Ub

Figure 4 | Regulation of the Fanconi protein complex in response to DNa damage and a model for interstrand cross link repair. a| S phase damage activates the ataxia telangiectasia and Rad3-related protein (ATR) checkpoint, Nature | Molecular Cell core Biology which phosphorylates several Fanconi anaemia proteins. The different Fanconi subunits are Reviews indicated: group I (the complex; shown in the rectangle) and group II (FANCI and FANCD2; the ID complex). FANCM and Fanconi anaemiaassociated protein of 24 kDa (FAAP24) have enzymatic activities. ATR-dependent phosphorylation of Fanconi anaemia proteins induces the ubiquitin ligase activity of the core complex and leads to robust ubiquitylation of FANCI and FANCD2. This is required for their association with chromatin in foci that contain several other recombination proteins: RAD51, RAD52, RAD54, and breast cancer type 1 susceptibility protein (BRCA1), as well as the FANCN (also known as PALB2), FANCJ (also known as BRIP1) and BRCA2 (also known as FANCD1) Fanconi subunits of group III (shown in blue). b| A model of interstrand cross link (ICL) repair, in which the replication forks (RFs) converge at the cross link. One leading strand is then extended to within one nucleotide of the ICL. The topological constraints are relieved by nicking events, which cause incisions and release the two sister strands. Repair then occurs through the translesion synthesis-mediated bypass of the lesion, followed by nucleotide excision repair-mediated excision of the lesion on one strand, and resection and homologous recombination repair of the other strand. ATRIP, ATR-interacting protein; Ub, ubiquitin.

Dicentric chromosome intermediate An unstable chromosome that has two centromeres.

DNA decatenation The unknotting of catenated structures.

Camptothecin A natural alkaloid that inhibits TOP1. Camptothecin analogues are often used in cancer chemotherapy.

RFs134 or by releasing the topological constraints caused by the ICl through incision events mediated by topoisomerases or specialized nucleases remains unknown. It is also possible that topoisomerases might need to collaborate with recombinases for the final resolution of the recombination structures formed during ICl repair. Interestingly, the Fanconi-associated factor BRCA1 was shown to promote DNA decatenation136. This is mediated through its interaction and colocalization with TOP2A (BOX 2), the ubiquitylation of which, mediated by the E3 ubiquitin ligase activity (BOX 3) of BRCA1, correlates with higher DNA decatenation activity 136. The replication checkpoint directly regulates the Fanconi anaemia pathway: both FANCD2 and FANCI are ATR targets137 and their phosphorylation by ATR affects the ubiquitylation of this complex (FIG. 4). A feedback loop between FANCM and the checkpoint effector kinase ChK1 (also known as ChEK1) (BOX 1) stabilizes these proteins from degradation by the proteasome under conditions of DNA damage138. Thus, a complex network of checkpoint and ubiquitin-mediated signalling and repair activities may be required to facilitate ICl repair and prevent the genomic instability associated with Fanconi anaemia.

DNA–protein cross links and DSB repair. DNA–protein complexes are induced by oxidative damage or uV irradiation. Anticancer camptothecins, such as topotecan or irinotecan, lead to reversible TOP1–DNA cleavage complexes, in which topoisomerases are covalently linked to the broken DNA strands139. It has been proposed that camptothecin treatment arrests advancing RFs through the accumulation of positive supercoils ahead of the replication machinery 140. Whether DSBs arise as a consequence of topological strain or the dissociation of the replisome when it encounters a topoisomerase–DNA complex (a nick) remains unknown. The repair of DSBs is not restricted to S phase and, therefore, an in-depth description of the mechanisms involved is not addressed here, but we invite readers to other reviews141,142. In addition to the DSBs formed by collisions of RFs with covalent DNA–protein complexes, RFs can encounter DSB sites or eroding telomeres. In a recent study in yeast it was shown that RFs irreversibly resolve at the break site, and that the Tel1–ATM checkpoint acts together with the Sae2 and the Mre11 nucleases to prevent the formation of cruciform structures at such terminal RFs75 (BOX 1). Arrest of RF movement on encountering a DSB has also been observed using an SV40 cell-free replication system,

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f o c u S o n G E n o m E I n SR ta b I lE IW ty EV S analysing the collision between a RF and a Top1-cleavable complex143. The termination of the RF that encounters the DSB does not impede the progression of the sister RF that is generated during replication initiation at the same replicon, suggesting that sister RFs can be uncoupled during replication75. A nearby DSB also triggers origin firing, even the firing of origins that are dormant under normal conditions of replication75.

Concluding remarks and perspectives Studies conducted in recent years have helped identify and establish the roles of several factors in activating the replication and damage checkpoint pathways following replication stress, promoting RF restart and progression or facilitating numerous replication-associated repair processes. Additional regulatory pathways involving ubiquitin and SuMO modifications now stand out, together with the regulatory pathways mediated by checkpoints and CDKs, as important coordinators of repair events and DNA transactions. how these factors and regulatory pathways affect topology and chromatin structure, in addition to influencing protein interactions, cellular distribution, or target stability, remain largely unsolved questions. Much has been learnt about checkpoints and their targets, as well as about other factors with important roles in replication and genome stability, such as RecQ proteins and DNA topoisomerases. however, many questions are arising and many still remain to be answered. Clearly, these fascinating enzymes will continue to keep scientists mesmerized and the replication and repair fields exciting. Schwob, E. Flexibility and governance in eukaryotic DNA replication. Curr. Opin. Microbiol. 7, 680–690 (2004). 2. Kearsey, S. E. & Cotterill, S. Enigmatic variations: divergent modes of regulating eukaryotic DNA replication. Mol. Cell 12, 1067–1075 (2003). 3. Zegerman, P. & Diffley, J. F. DNA replication as a target of the DNA damage checkpoint. DNA Repair (Amst.) 8, 1077–1088 (2009). 4. Raghuraman, M. K. et al. Replication dynamics of the yeast genome. Science 294, 115–121 (2001). 5. Yabuki, N., Terashima, H. & Kitada, K. Mapping of early firing origins on a replication profile of budding yeast. Genes Cells 7, 781–789 (2002). 6. Wyrick, J. J. et al. Genome‑wide distribution of ORC and MCM proteins in S. cerevisiae: high‑resolution mapping of replication origins. Science 294, 2357–2360 (2001). References 5 and 6 map replication origins and identify their replication time. 7. Dershowitz, A. & Newlon, C. S. The effect on chromosome stability of deleting replication origins. Mol. Cell. Biol. 13, 391–398 (1993). Analyzes the consequences of ablating origins and suggests that origins are present in excess. 8. Woodward, A. M. et al. Excess Mcm2–7 license dormant origins of replication that can be used under conditions of replicative stress. J. Cell Biol. 173, 673–683 (2006). 9. Ibarra, A., Schwob, E. & Mendez, J. Excess MCM proteins protect human cells from replicative stress by licensing backup origins of replication. Proc. Natl Acad. Sci. USA 105, 8956–8961 (2008). 10. Okuno, Y., McNairn, A. J., den Elzen, N., Pines, J. & Gilbert, D. M. Stability, chromatin association and functional activity of mammalian pre‑replication complex proteins during the cell cycle. EMBO J. 20, 4263–4277 (2001). 11. Laskey, R. A. & Harland, R. M. Replication origins in the eukaryotic chromosome. Cell 24, 283–284 (1981). 12. Ivessa, A. S. et al. The Saccharomyces cerevisiae helicase Rrm3p facilitates replication past nonhistone 1.

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Genetic screens in yeasts and vertebrates and enzymological studies have been extended and combined with molecular and cellular biology approaches in an attempt to delineate the underlying causes of genome instability that are triggered by different types of replication stress or mutations in replication factors. Such causes include altered replication kinetics, the formation of unusual DNA structures, defects in the recruitment of appropriate DNA repair factors, unusual RF restart mechanisms and unleashed alternative pathways inducing chromosomal aberrations — the mechanisms are too many to be listed here in their entirety. how DNA topology or genome architecture influence repair, replication or the expression of fragile zones, is an emerging topic, the importance of which is already being appreciated. Importantly, a linkage between replication anomalies and the cancer phenotype is now clear. The knowledge derived from understanding the mechanisms that induce or suppress replication problems from propagating into genome instability, therefore, impacts on our understanding of cancer and the design of cancer therapies. Checkpoint and topoisomerase inhibitors are examples of some of the most widely used drugs for treating tumours. The insights and conceptual frameworks derived from genetic studies conducted in yeast and vertebrate cells, aimed at identifying synthetic lethal interactions or at understanding the pathways responding to different drugs, should help delineate the rationale for chemotherapeutic combinations that will reduce the cancer burden.

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Acknowledgements

We thank all members of our laboratories for helpful discus‑ sions. The work in D.B’s laboratory is supported by the European Research Council grant 242928 and the Associazione Italiana per la Ricerca sul Cancro. The work in M.F’s laboratory is supported by grants from Telethon, the Associazione Italiana per la Ricerca sul Cancro and the European Community.

Competing interests statement

The authors declare no competing financial interests.

DATABASES uniProtKb: http://www.uniprot.org ATM | ATR | BLM | BRCA1 | BRCA2 | Cdc6 | Cdc7 | Cdc45 | Csm3 | Dbf4 | DinB | FAAP24 | FANCD2 | FANCI | FANCJ | FANCM | FANCN | Hmo1 | Mec1 | Mms21 | Mph1 | Mrc1 | PCNA | Pmt3 | Rad51 | Rad53 | Rtf2 | Sgs1 | Smt3 | Tel1 | Tof1 | Top1 | Top2 | Ubc9

FURTHER INFORMATION Dana branzei’s homepage: http://www.ifom-ieo-campus.it/ research/branzei.php marco foiani’s homepage: http://www.ifom-ieo-campus.it/ research/foiani.php all liNks aRe acTive iN The oNliNe pDF

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