Impediments to DNA replication are known to induce gross chromosomal rearrangements (GCR) and copy number variations (CNV). GCRs/CNVs underlie human genomic disorders1 and are a feature of cancer2. During cancer development environmental factors and oncogene-driven proliferation promote replication stress. Resulting GCRs/CNVs are proposed to contribute to cancer development and therapy resistance3. When stress arrests replication, the replisome remains associated with the fork DNA (stalled fork) and is protected by the inter-S phase checkpoint. Stalled forks efficiently resume when the stress is relieved. However, if the polymerases dissociate from the fork (fork collapse) or the fork structure breaks (broken fork), replication restart can proceed either by homologous recombination (HR) or microhomology-primed re-initiation (FoSTeS/MMBIR)4,5. Here we ascertain the consequences of replication with a fork restarted by HR. We identify a new mechanism of chromosomal rearrangement: recombination-restarted forks have an exceptionally high propensity to execute a U-turn at small inverted repeats (up to 1:40 replication events). We propose that the error-prone nature of restarted forks contributes to the generation of GCRs and gene amplification in cancer and to non-recurrent CNVs in genomic disorders.
The replication checkpoint is a dedicated sensor-response system activated by impeded replication forks. It stabilizes stalled forks and arrests division, thereby preserving genome integrity and promoting cell survival. In budding yeast, Tof1 is thought to act as a specific mediator of the replication checkpoint signal that activates the effector kinase Rad53. Here we report studies of fission yeast Swi1, a Tof1-related protein required for a programmed fork-pausing event necessary for mating type switching. Our studies have shown that Swi1 is vital for proficient activation of the Rad53-like checkpoint kinase Cds1. Together they are required to prevent fork collapse in the ribosomal DNA repeats, and they also prevent irreversible fork arrest at a newly identified hydroxyurea pause site. Swi1 also has Cds1-independent functions. Rad22 DNA repair foci form during S phase in swi1 mutants and to a lesser extent in cds1 mutants, indicative of fork collapse. Mus81, a DNA endonuclease required for recovery from collapsed forks, is vital in swi1 but not cds1 mutants. Swi1 is recruited to chromatin during S phase. We propose that Swi1 stabilizes replication forks in a configuration that is recognized by replication checkpoint sensors.
The minichromosome maintenance (MCM) complex plays essential, conserved roles throughout DNA synthesis: first, as a component of the prereplication complex at origins and, then, as a helicase associated with replication forks. Here we use fission yeast (Schizosaccharomyces pombe) as a model to demonstrate a role for the MCM complex in protecting replication fork structure and promoting recovery from replication arrest. Loss of MCM function generates lethal double-strand breaks at sites of DNA synthesis during replication elongation, suggesting replication fork collapse. MCM function also maintains the stability of forks stalled by hydroxyurea that activate the replication checkpoint. In cells where the checkpoint is activated, Mcm4 binds the Cds1 kinase and undergoes Cds1-dependent phosphorylation. MCM proteins also interact with proteins involved in homologous recombination, which promotes recovery from arrest by ensuring normal mitosis. We suggest that the MCM complex links replication fork stabilization with checkpoint arrest and recovery through direct interactions with checkpoint and recombination proteins and that this role in S-phase genome stability is conserved from yeast to human cells.
Swi1 is required for programmed pausing of replication forks near the mat1 locus in the fission yeast Schizosaccharomyces pombe. This fork pausing is required to initiate a recombination event that switches mating type. Swi1 is also needed for the replication checkpoint that arrests division in response to fork arrest. How Swi1 accomplishes these tasks is unknown. Here we report that Swi1 copurifies with a 181-amino-acid protein encoded by swi3+. The Swi1-Swi3 complex is required for survival of fork arrest and for activation of the replication checkpoint kinase Cds1. Association of Swi1 and Swi3 with chromatin during DNA replication correlated with movement of the replication fork. swi1Δ and swi3Δ mutants accumulated Rad22 (Rad52 homolog) DNA repair foci during replication. These foci correlated with the Rad22-dependent appearance of Holliday junction (HJ)-like structures in cells lacking Mus81-Eme1 HJ resolvase. Rhp51 and Rhp54 homologous recombination proteins were not required for viability in swi1Δ or swi3Δ cells, indicating that the HJ-like structures arise from single-strand DNA gaps or rearranged forks instead of broken forks. We propose that Swi1 and Swi3 define a fork protection complex that coordinates leading- and lagging-strand synthesis and stabilizes stalled replication forks.
In response to DNA damage and replication pausing, eukaryotes activate checkpoint pathways that prevent genomic instability by coordinating cell cycle progression with DNA repair. The intra-S-phase checkpoint has been proposed to protect stalled replication forks from pathological rearrangements that could result from unscheduled recombination. On the other hand, recombination may be needed to cope with either stalled forks or double-strand breaks resulting from hydroxyurea treatment. We have exploited fission yeast to elucidate the relationship between replication fork stalling, loading of replication and recombination proteins onto DNA, and the intra-S checkpoint. Here, we show that a functional recombination machinery is not essential for recovery from replication fork arrest and instead can lead to nonfunctional fork structures. We find that Rad22-containing foci are rare in S-phase cells, but peak in G2 phase cells after a perturbed S phase. Importantly, we find that the intra-S checkpoint is necessary to avoid aberrant strand-exchange events during a hydroxyurea block.
Faithful DNA replication is essential to all life. Hydroxyurea (HU) depletes the cells of dNTPs, which initially results in stalled replication forks that, after prolonged treatment, collapse into DSBs. Here, we report that stalled replication forks are efficiently restarted in a RAD51-dependent process that does not trigger homologous recombination (HR). The XRCC3 protein, which is required for RAD51 foci formation, is also required for replication restart of HU-stalled forks, suggesting that RAD51-mediated strand invasion supports fork restart. In contrast, replication forks collapsed by prolonged replication blocks do not restart, and global replication is rescued by new origin firing. We find that RAD51-dependent HR is triggered for repair of collapsed replication forks, without apparent restart. In conclusion, our data suggest that restart of stalled replication forks and HR repair of collapsed replication forks require two distinct RAD51-mediated pathways.
► RAD51 promotes replication fork restart after short hydroxyurea (HU) blocks ► RAD51-mediated fork restart does not create homologous recombination (HR) products ► Long HU blocks lead to fork collapse; replication is restarted by new origin firing ► Collapsed replication forks are repaired by classical RAD51-dependent HR
Chromosome breakage as a result of replication stress has been hypothesized to be the direct consequence of defective replication fork progression, or “collapsed” replication forks. However, direct and genome-wide evidence that collapsed replication forks give rise to chromosome breakage is still lacking. Previously we showed that a yeast replication checkpoint mutant mec1-1, after transient exposure to replication impediment imposed by hydroxyurea (HU), failed to complete DNA replication, accumulated single-stranded DNA (ssDNA) at the replication forks, and fragmented its chromosomes. In this study, by following replication fork progression genome-wide via ssDNA detection and by direct mapping of chromosome breakage after HU exposure, we have tested the hypothesis that the chromosome breakage in mec1 cells occurs at collapsed replication forks. We demonstrate that sites of chromosome breakage indeed correlate with replication fork locations. Moreover, ssDNA can be detected prior to chromosome breakage, suggesting that ssDNA accumulation is the common precursor to double strand breaks at collapsed replication forks.
chromosome fragile sites; double strand breaks; mec1; replication checkpoint; single-stranded DNA
Replication fork arrest is a recognized source of genetic instability, and transcription is one of the most prominent causes of replication impediment. We analyze here the requirement for recombination proteins in Escherichia coli when replication–transcription head-on collisions are induced at a specific site by the inversion of a highly expressed ribosomal operon (rrn). RecBC is the only recombination protein required for cell viability under these conditions of increased replication-transcription collisions. In its absence, fork breakage occurs at the site of collision, and the resulting linear DNA is not repaired and is slowly degraded by the RecJ exonuclease. Lethal fork breakage is also observed in cells that lack RecA and RecD, i.e. when both homologous recombination and the potent exonuclease V activity of the RecBCD complex are inactivated, with a slow degradation of the resulting linear DNA by the combined action of the RecBC helicase and the RecJ exonuclease. The sizes of the major linear fragments indicate that DNA degradation is slowed down by the encounter with another rrn operon. The amount of linear DNA decreases nearly two-fold when the Holliday junction resolvase RuvABC is inactivated in recB, as well as in recA recD mutants, indicating that part of the linear DNA is formed by resolution of a Holliday junction. Our results suggest that replication fork reversal occurs after replication–transcription head-on collision, and we propose that it promotes the action of the accessory replicative helicases that dislodge the obstacle.
Genomes are duplicated prior to cell division by DNA replication, and in all organisms replication impairment leads to chromosome instability. In bacteria, replication and transcription take place simultaneously, and in eukaryotes house-keeping genes are expressed during the S-phase; consequently, transcription is susceptible to impair replication progression. Here, we increase head-on replication–transcription collisions on the bacterial chromosome by inversion of a ribosomal operon (rrn). We show that only one recombination protein is required for growth when the rrn genes are highly expressed: the RecBCD complex, an exonuclease/recombinase that promotes degradation and RecA-dependent homologous recombination of linear DNA. In the absence of RecBCD, we observe linear DNA that ends in the collision region. This linear DNA is composed of only the origin-proximal region of the inverted rrn operon, indicating that it results from fork breakage. It is partly RuvABC-dependent (i.e. produced by the E. coli Holliday junction resolvase), indicating that blocked forks are reversed. The linear DNA ends up at the inverted rrn locus only if the RecJ exonuclease is inactivated; otherwise it is degraded, with major products ending in other upstream rrn operons, indicating that DNA degradation is slowed down by ribosomal operon sequences.
High-fidelity replication of DNA, and its accurate segregation to daughter cells, is critical for maintaining genome stability and suppressing cancer. DNA replication forks are stalled by many DNA lesions, activating checkpoint proteins that stabilize stalled forks. Stalled forks may eventually collapse, producing a broken DNA end. Fork restart is typically mediated by proteins initially identified by their roles in homologous recombination repair of DNA double-strand breaks (DSBs). In recent years, several proteins involved in DSB repair by non-homologous end joining (NHEJ) have been implicated in the replication stress response, including DNA-PKcs, Ku, DNA Ligase IV-XRCC4, Artemis, XLF and Metnase. It is currently unclear whether NHEJ proteins are involved in the replication stress response through indirect (signaling) roles, and/or direct roles involving DNA end joining. Additional complexity in the replication stress response centers around RPA, which undergoes significant post-translational modification after stress, and RAD52, a conserved HR protein whose role in DSB repair may have shifted to another protein in higher eukaryotes, such as BRCA2, but retained its role in fork restart. Most cancer therapeutic strategies create DNA replication stress. Thus, it is imperative to gain a better understanding of replication stress response proteins and pathways to improve cancer therapy.
DNA repair; genome instability; cancer therapy
Replication forks stall at DNA lesions or as a result of an unfavorable replicative environment. These fork stalling events have been associated with recombination and gross chromosomal rearrangements. Recombination and fork bypass pathways are the mechanisms accountable for restart of stalled forks. An important lesion bypass mechanism is the highly conserved post-replication repair (PRR) pathway that is composed of error-prone translesion and error-free bypass branches. EXO1 codes for a Rad2p family member nuclease that has been implicated in a multitude of eukaryotic DNA metabolic pathways that include DNA repair, recombination, replication, and telomere integrity. In this report, we show EXO1 functions in the MMS2 error-free branch of the PRR pathway independent of the role of EXO1 in DNA mismatch repair (MMR). Consistent with the idea that EXO1 functions independently in two separate pathways, we defined a domain of Exo1p required for PRR distinct from those required for interaction with MMR proteins. We then generated a point mutant exo1 allele that was defective for the function of Exo1p in MMR due to disrupted interaction with Mlh1p, but still functional for PRR. Lastly, by using a compound exo1 mutant that was defective for interaction with Mlh1p and deficient for nuclease activity, we provide further evidence that Exo1p plays both structural and catalytic roles during MMR.
DNA mismatch repair; EXO1; DNA nuclease; DNA damage and post-replication repair
Elaborate processes act at the DNA replication fork to minimize the generation of chromatid discontinuity when lesions are encountered. To prevent collapse of stalled replication forks, mutagenic translesion synthesis (TLS) polymerases are recruited temporarily to bypass DNA lesions. When a replication-associated (one-ended) double strand break occurs, homologous recombination repair (HRR) can restore chromatid continuity in what has traditionally been regarded as an “error-free” process. Our previous mutagenesis studies show an important role for HRR in preventing deletions and rearrangements that would otherwise result from error-prone nonhomologous end joining (NHEJ) after fork breakage. An analogous, but distinct, role in minimizing mutations is attributed to the proteins defective in the cancer predisposition disease Fanconi anemia (FA). Cells from FA patients and model systems show an increased proportion of gene-disrupting deletions at the hprt locus as well as decreased mutation rates in the hprt assay, suggesting a role for the FANC proteins in promoting TLS, HRR, and possibly also NHEJ. It remains unclear whether HRR, like the FANC pathway, impacts the rate of base substitution mutagenesis. Therefore, we measured, in isogenic rad51d and fancg CHO mutants, mutation rates at the Na+/K+–ATPase α-subunit (ATP1A1) locus using ouabain resistance, which specifically detects base substitution mutations. Surprisingly, we found that the spontaneous mutation rate was reduced ~2.5-fold in rad51d knockout cells, an even greater extent than observed in fancg cells, when compared with parental and isogenic gene-complemented control lines. A ~2-fold reduction in induced mutations in rad51d cells was seen after treatment with the DNA alkylating agent ethylnitrosurea while a lesser reduction occurred in fancg cells. Should the model ATP1A1 locus be representative of the genome, we conclude that at least 50% of base substitution mutations in this mammalian system arise through error-prone polymerase(s) acting during HRR-mediated restart of broken replication forks.
Fanconi anemia; homologous recombination; translesion synthesis; CHO cells; ouabain resistance
Homologous recombination (HR) is a major mechanism utilized to repair blockage of DNA replication forks. Here, we report that a sister chromatid exchange (SCE) generated by crossover-associated HR efficiently occurs in response to replication fork stalling before any measurable DNA double-strand breaks (DSBs). Interestingly, SCE produced by replication fork collapse following DNA DSBs creation is specifically suppressed by ATR, a central regulator of the replication checkpoint. BRCA1 depletion leads to decreased RPA2 phosphorylation (RPA2-P) following replication fork stalling but has no obvious effect on RPA2-P following replication fork collapse. Importantly, we found that BRCA1 promotes RAD51 recruitment and SCE induced by replication fork stalling independent of ATR. In contrast, BRCA1 depletion leads to a more profound defect in RAD51 recruitment and SCE induced by replication fork collapse when ATR is depleted. We concluded that BRCA1 plays a dual role in two distinct HR-mediated repair upon replication fork stalling and collapse. Our data established a molecular basis for the observation that defective BRCA1 leads to a high sensitivity to agents that cause replication blocks without being associated with DSBs, and also implicate a novel mechanism by which loss of cell cycle checkpoints promotes BRCA1-associated tumorigenesis via enhancing HR defect resulting from BRCA1 deficiency.
Genome duplication requires that replication forks track the entire length of every chromosome. When complications occur, homologous recombination-mediated repair supports replication fork movement and recovery. This leads to physical connections between the nascent sister chromatids in the form of Holliday junctions and other branched DNA intermediates. A key role in the removal of these recombination intermediates falls to structure-specific nucleases such as the Holliday junction resolvase RuvC in Escherichia coli. RuvC is also known to cut branched DNA intermediates that originate directly from blocked replication forks, targeting them for origin-independent replication restart. In eukaryotes, multiple structure-specific nucleases, including Mus81–Mms4/MUS81–EME1, Yen1/GEN1, and Slx1–Slx4/SLX1–SLX4 (FANCP) have been implicated in the resolution of branched DNA intermediates. It is becoming increasingly clear that, as a group, they reflect the dual function of RuvC in cleaving recombination intermediates and failing replication forks to assist the DNA replication process.
In fission yeast, replication fork arrest activates the replication checkpoint effector kinase Cds1Chk2/Rad53 through the Rad3ATR/Mec1-Mrc1Claspin pathway. Hsk1, the Cdc7 homolog of fission yeast required for efficient initiation of DNA replication, is also required for Cds1 activation. Hsk1 kinase activity is required for induction and maintenance of Mrc1 hyperphosphorylation, which is induced by replication fork block and mediated by Rad3. Rad3 kinase activity does not change in an hsk1 temperature-sensitive mutant, and Hsk1 kinase activity is not affected by rad3 mutation. Hsk1 kinase vigorously phosphorylates Mrc1 in vitro, predominantly at non-SQ/TQ sites, but this phosphorylation does not seem to affect the Rad3 action on Mrc1. Interestingly, the replication stress-induced activation of Cds1 and hyperphosphorylation of Mrc1 is almost completely abrogated in an initiation-defective mutant of cdc45, but not significantly in an mcm2 or polε mutant. These results suggest that Hsk1-mediated loading of Cdc45 onto replication origins may play important roles in replication stress-induced checkpoint.
Cdc7; Cdc45; checkpoint; DNA replication; Mrc1
Failure to reactivate stalled or collapsed DNA replication forks is a potential source of genomic instability. Homologous recombination (HR) is a major mechanism for repairing the DNA damage resulting from replication arrest. The single-strand DNA (ssDNA)-binding protein, replication protein A (RPA), plays a major role in multiple processes of DNA metabolism. However, the role of RPA2 hyperphosphorylation, which occurs in response to DNA damage, had been unclear. Here, we show that hyperphosphorylated RPA2 associates with ssDNA and recombinase protein Rad51 in response to replication arrest by hydroxyurea (HU) treatment. In addition, RPA2 hyperphosphorylation is critical for Rad51 recruitment and HR-mediated repair following HU. However, RPA2 hyperphosphorylation is not essential for both ionizing radiation (IR)-induced Rad51 foci formation and I-Sce-I endonuclease-stimulated HR. Moreover, we show that expression of a phosphorylation-deficient mutant of RPA2 leads to increased chromosomal aberrations following HU treatment but not after exposure to IR. Finally, we demonstrate that loss of RPA2 hyperphosphorylation results in a loss of viability when cells are confronted with replication stress whereas cells expressing hyperphosphorylation-defective RPA2 or wild-type RPA2 have a similar sensitivity to IR. Thus, our data suggest that RPA2 hyperphosphorylation plays a critical role in maintenance of genomic stability and cell survival after a DNA replication block via promotion of HR.
Maintaining the stability of the replication forks is one of the main tasks of the DNA damage response. Specifically, checkpoint mechanisms detect stressed forks and prevent their collapse. In the published report reviewed here we have shown that defective chromatin assembly in cells lacking either H3K56 acetylation or the chromatin assembly factors CAF1 and Rtt106 affects the integrity of advancing replication forks, despite the presence of functional checkpoints. This loss of replication intermediates is exacerbated in the absence of Rad52, suggesting that collapsed forks are rescued by homologous recombination and providing an explanation for the accumulation of recombinogenic DNA damage displayed by these mutants. These phenotypes mimic those obtained by a partial reduction in the pool of available histones and are consistent with a model in which defective histone deposition uncouples DNA synthesis and nucleosome assembly, thus making the fork more susceptible to collapse. Here, we review these findings and discuss the possibility that defects in the lagging strand represent a major source of fork instability in chromatin assembly mutants.
Asf1; H3K56 acetylation; Okazaki fragment; Rad27; genetic instability; homologous recombination; nucleosome assembly; replication fork
DNA damage checkpoints coordinate the cellular response to genotoxic stress and arrest the cell cycle in response to DNA damage and replication fork stalling. Homologous recombination is a ubiquitous pathway for the repair of DNA double-stranded breaks and other checkpoint-inducing lesions. Moreover, homologous recombination is involved in postreplicative tolerance of DNA damage and the recovery of DNA replication after replication fork stalling. Here, we show that the phosphorylation on serines 2, 8, and 14 (S2,8,14) of the Rad55 protein is specifically required for survival as well as for normal growth under genome-wide genotoxic stress. Rad55 is a Rad51 paralog in Saccharomyces cerevisiae and functions in the assembly of the Rad51 filament, a central intermediate in recombinational DNA repair. Phosphorylation-defective rad55-S2,8,14A mutants display a very slow traversal of S phase under DNA-damaging conditions, which is likely due to the slower recovery of stalled replication forks or the slower repair of replication-associated DNA damage. These results suggest that Rad55-S2,8,14 phosphorylation activates recombinational repair, allowing for faster recovery after genotoxic stress.
DNA damage that blocks replication is bypassed in order to complete chromosome duplication and preserve cell viability and genome stability. Rad5, a PCNA polyubiquitin ligase and DNA-dependent ATPase in yeast, is orthologous to putative tumor suppressors and controls error-free damage bypass by an unknown mechanism. To identify the mechanism in vivo, we investigated the roles of Rad5 and analyzed the DNA structures that form during damage bypass at site-specific stalled forks present at replication origins. Rad5 mediated the formation of recombination-dependent, X-shaped DNA structures containing Holliday junctions between sister chromatids. Mutants lacking these damage-induced chromatid junctions were defective in resolving stalled forks, restarting replication and completing chromosome duplication. Rad5 polyubiquitin ligase and ATPase domains both contributed to replication fork recombination. Our results indicate that multiple activities of Rad5 function coordinately with homologous recombination factors to enable replication template switch events that join sister chromatids at stalled forks and bypass DNA damage.
Replication fork reversal (RFR) is a reaction that takes place in Escherichia coli at replication forks arrested by the inactivation of a replication protein. Fork reversal involves the annealing of the leading and lagging strand ends; it results in the formation of a Holliday junction adjacent to DNA double-strand end, both of which are processed by recombination enzymes. In several replication mutants, replication fork reversal is catalysed by the RuvAB complex, originally characterized for its role in the last steps of homologous recombination, branch migration and resolution of Holliday junctions. We present here the isolation and characterization of ruvA and ruvB single mutants that are impaired for RFR at forks arrested by the inactivation of polymerase III, while they remain capable of homologous recombination. The positions of the mutations in the proteins and the genetic properties of the mutants suggest that the mutations affect DNA binding, RuvA–RuvB interaction and/or RuvB-helicase activity. These results show that a partial RuvA or RuvB defect affects primarily RFR, implying that RFR is a more demanding reaction than Holliday junction resolution.
Genome integrity is jeopardized each time DNA replication forks stall or collapse. Here, we report the identification of a complex composed of MMS22L (C6ORF167) and TONSL (NFKBIL2) that participates in the recovery from replication stress. MMS22L and TONSL are homologous to yeast Mms22 and plant Tonsoku/Brushy1, respectively. MMS22L-TONSL accumulates at regions of ssDNA associated with distressed replication forks or at processed DNA breaks, and its depletion results in high levels of endogenous DNA double-strand breaks caused by an inability to complete DNA synthesis after replication fork collapse. Moreover, cells depleted of MMS22L are highly sensitive to camptothecin, a topoisomerase I poison that impairs DNA replication progression. Finally, MMS22L and TONSL are necessary for the efficient formation of RAD51 foci after DNA damage and their depletion impairs homologous recombination. These results indicate that MMS22L and TONSL are genome caretakers that stimulate the recombination-dependent repair of stalled or collapsed replication forks.
PMID: 21055983 CAMSID: cams1662
DNA double-strand breaks; DNA replication; Homologous recombination; siRNA; RAD51; camptothecin
The replication machinery, or the replisome, collides with a variety of obstacles during the normal process of DNA replication. In addition to damaged template DNA, numerous chromosome regions are considered to be difficult to replicate owing to the presence of DNA secondary structures and DNA-binding proteins. Under these conditions, the replication fork stalls, generating replication stress. Stalled forks are prone to collapse, posing serious threats to genomic integrity. It is generally thought that the replication checkpoint functions to stabilize the replisome and replication fork structure upon replication stress. This is important in order to allow DNA replication to resume once the problem is solved. However, our recent studies demonstrated that some replisome components undergo proteasome-dependent degradation during DNA replication in the fission yeast Schizosaccharomyces pombe. Our investigation has revealed the involvement of the SCFPof3 (Skp1-Cullin/Cdc53-F-box) ubiquitin ligase in replisome regulation. We also demonstrated that forced accumulation of the replisome components leads to abnormal DNA replication upon replication stress. Here we review these findings and present additional data indicating the importance of replisome degradation for DNA replication. Our studies suggest that cells activate an alternative pathway to degrade replisome components in order to preserve genomic integrity.
DNA replication; replisome; protein degradation; proteasome; ubiquitin ligase; F-box; replication fork protection complex; FPC
Homologous Recombination (HR) is an essential genome stability mechanism used for high-fidelity repair of DNA double-strand breaks and for the recovery of stalled or collapsed DNA replication forks. The crucial homology search and DNA strand exchange steps of HR are catalyzed by presynaptic filaments—helical filaments of a recombinase enzyme bound to single-stranded DNA. Presynaptic filaments are fundamentally dynamic structures, the assembly, catalytic turnover, and disassembly of which must be closely coordinated with other elements of the DNA recombination, repair, and replication machinery in order for genome maintenance functions to be effective. Here, we review the major dynamic elements controlling the assembly, activity, and disassembly of presynaptic filaments: some intrinsic such as recombinase ATP binding and hydrolytic activities, others extrinsic such as ssDNA-binding proteins, mediator proteins, and DNA motor proteins. We examine dynamic behavior on multiple levels, including atomic- and filament-level structural changes associated with ATP binding and hydrolysis as evidenced in crystal structures, as well as subunit binding and dissociation events driven by intrinsic and extrinsic factors. We examine the biochemical properties of recombination proteins from four model systems (T4 phage, E. coli, S. cerevisiae, and H. sapiens), demonstrating how their properties are tailored for the context-specific requirements in these diverse species. We propose that the presynaptic filament has evolved to rely on multiple external factors for increased multi-level regulation of HR processes in genomes with greater structural and sequence complexity.
recombinase; RecA; Rad51; BRCA2; mediator proteins; SSB; helicase; translocase
Cleavage of a DNA replication fork leads to fork restoration by recombination repair. In prokaryote cells carrying restriction–modification systems, fork passage reduces genome methylation by the modification enzyme and exposes the chromosome to attack by the restriction enzyme. Various observations have suggested a relationship between the fork and Type I restriction enzymes, which cleave DNA at a distance from a recognition sequence. Here, we demonstrate that a Type I restriction enzyme preparation cleaves a model replication fork at its branch. The enzyme probably tracks along the DNA from an unmethylated recognition site on the daughter DNA and cuts the fork upon encountering the branch point. Our finding suggests that these restriction–modification systems contribute to genome maintenance through cell death and indicates that DNA replication fork cleavage represents a critical point in genome maintenance to choose between the restoration pathway and the destruction pathway.
DNA replication initiated by one-ended homologous recombination at a double-strand break is highly inaccurate, as it greatly stimulates frameshift mutations over the entire path of the replication fork.
DNA must be synthesized for purposes of genome duplication and DNA repair. While the former is a highly accurate process, short-patch synthesis associated with repair of DNA damage is often error-prone. Break-induced replication (BIR) is a unique cellular process that mimics normal DNA replication in its processivity, rate, and capacity to duplicate hundreds of kilobases, but is initiated at double-strand breaks (DSBs) rather than at replication origins. Here we employed a series of frameshift reporters to measure mutagenesis associated with BIR in Saccharomyces cerevisiae. We demonstrate that BIR DNA synthesis is intrinsically inaccurate over the entire path of the replication fork, as the rate of frameshift mutagenesis during BIR is up to 2,800-fold higher than during normal replication. Importantly, this high rate of mutagenesis was observed not only close to the DSB where BIR is less stable, but also far from the DSB where the BIR replication fork is fast and stabilized. We established that polymerase proofreading and mismatch repair correct BIR errors. Also, dNTP levels were elevated during BIR, and this contributed to BIR-related mutagenesis. We propose that a high level of DNA polymerase errors that is not fully compensated by error-correction mechanisms is largely responsible for mutagenesis during BIR, with Pol δ generating many of the mutagenic errors. We further postulate that activation of BIR in eukaryotic cells may significantly contribute to accumulation of mutations that fuel cancer and evolution.
Accurate transmission of genetic information requires the precise replication of parental DNA. Mutations (which can be beneficial or deleterious) arise from errors that remain uncorrected. DNA replication occurs during S-phase of the cell cycle and is extremely accurate due to highly selective DNA polymerases coupled with effective error-correction mechanisms. In contrast, DNA synthesis associated with short-patch DNA repair is often error-prone. Break-induced replication (BIR) presents an interesting case of large-scale DNA duplication that occurs in the context of DNA repair. In this study we employed a yeast-based system to investigate the level of mutagenesis associated with BIR compared to mutagenesis during normal DNA replication. We report that frameshifts, which are the most deleterious kind of point mutation, are much more frequent during BIR than during normal DNA replication. Surprisingly, we observed that the majority of mutations associated with BIR were created by polymerases responsible for normal DNA replication, which are assumed to be highly precise. Overall, we propose that BIR is a novel source of mutagenesis that may contribute to disease genesis and evolution.
Protection of genome integrity depends on the coordinated activities of DNA replication, DNA repair, chromatin assembly and chromosome segregation mechanisms. DNA lesions are detected by the master checkpoint kinases ATM (Tel1) and ATR (Rad3/Mec1), which phosphorylate multiple substrates, including a C-terminal SQ motif in histone H2A or H2AX. The 6-BRCT domain protein Brc1, which is required for efficient recovery from replication fork arrest and collapse in fission yeast, binds phospho-histone H2A (γH2A)-coated chromatin at stalled and damaged replication forks. We recently found that Brc1 co-localizes with γH2A that appears in pericentromeric heterochromatin during S-phase. Our studies indicate that Brc1 contributes to the maintenance of pericentromeric heterochromatin, which is required for efficient chromosome segregation during mitosis. Here, we review these studies and present additional results that establish the functional requirements for the N-terminal BRCT domains of Brc1 in the replication stress response and resistance to the microtubule destabilizing drug thiabendazole (TBZ). We also identify the nuclear localization signal (NLS) in Brc1, which closely abuts the C-terminal pair of BRCT domains that form the γH2A-binding pocket. This compact arrangement of localization domains may be a shared feature of other γH2A-binding proteins, including Rtt107, PTIP and Mdc1.
DNA damage response; replication stress; heterochromatin; centromere; BRCT domain; mitosis; nuclear localization signal