The ZGRF1 Helicase Promotes Recombinational Repair of Replication-Blocking DNA Damage in Human Cells.

Replication-blocking DNA lesions are particularly toxic to proliferating cells because they can lead to chromosome mis-segregation if not repaired prior to mitosis. In this study, we report that ZGRF1 null cells accumulate chromosome aberrations following replication perturbation and show sensitivity to two potent replication-blocking anticancer drugs: mitomycin C and camptothecin. Moreover, ZGRF1 null cells are defective in catalyzing DNA damage-induced sister chromatid exchange despite accumulating excessive FANCD2, RAD51, and γ-H2AX foci upon induction of interstrand DNA crosslinks. Consistent with a direct role in promoting recombinational DNA repair, we show that ZGRF1 is a 5'-to-3' helicase that catalyzes D-loop dissociation and Holliday junction branch migration. Moreover, ZGRF1 physically interacts with RAD51 and stimulates strand exchange catalyzed by RAD51-RAD54. On the basis of these data, we propose that ZGRF1 promotes repair of replication-blocking DNA lesions through stimulation of homologous recombination.

Esc2 promotes telomere stability in response to DNA replication stress.

Telomeric regions of the genome are inherently difficult-to-replicate due to their propensity to generate DNA secondary structures and form nucleoprotein complexes that can impede DNA replication fork progression. Precisely how cells respond to DNA replication stalling within a telomere remains poorly characterized, largely due to the methodological difficulties in analysing defined stalling events in molecular detail. Here, we utilized a site-specific DNA replication barrier mediated by the 'Tus/Ter' system to define the consequences of DNA replication perturbation within a single telomeric locus. Through molecular genetic analysis of this defined fork-stalling event, coupled with the use of a genome-wide genetic screen, we identified an important role for the SUMO-like domain protein, Esc2, in limiting genome rearrangements at a telomere. Moreover, we showed that these rearrangements are driven by the combined action of the Mph1 helicase and the homologous recombination machinery. Our findings demonstrate that chromosomal context influences cellular responses to a stalled replication fork and reveal protective factors that are required at telomeric loci to limit DNA replication stress-induced chromosomal instability.

A Molecular Toolbox to Engineer Site-Specific DNA Replication Perturbation.

Site-specific arrest of DNA replication is a useful tool for analyzing cellular responses to DNA replication perturbation. The E. coli Tus-Ter replication barrier can be reconstituted in eukaryotic cells as a system to engineer an unscheduled collision between a replication fork and an "alien" impediment to DNA replication. To further develop this system as a versatile tool, we describe a set of reagents and a detailed protocol that can be used to engineer Tus-Ter barriers into any locus in the budding yeast genome. Because the Tus-Ter complex is a bipartite system with intrinsic DNA replication-blocking activity, the reagents and protocols developed and validated in yeast could also be optimized to engineer site-specific replication fork barriers into other eukaryotic cell types.

Stalled replication forks generate a distinct mutational signature in yeast.

Proliferating cells acquire genome alterations during the act of DNA replication. This leads to mutation accumulation and somatic cell mosaicism in multicellular organisms, and is also implicated as an underlying cause of aging and tumorigenesis. The molecular mechanisms of DNA replication-associated genome rearrangements are poorly understood, largely due to methodological difficulties in analyzing specific replication forks in vivo. To provide an insight into this process, we analyzed the mutagenic consequences of replication fork stalling at a single, site-specific replication barrier (the Escherichia coli Tus/Ter complex) engineered into the yeast genome. We demonstrate that transient stalling at this barrier induces a distinct pattern of genome rearrangements in the newly replicated region behind the stalled fork, which primarily consist of localized losses and duplications of DNA sequences. These genetic alterations arise through the aberrant repair of a single-stranded DNA gap, in a process that is dependent on Exo1- and Shu1-dependent homologous recombination repair (HRR). Furthermore, aberrant processing of HRR intermediates, and elevated HRR-associated mutagenesis, is detectable in a yeast model of the human cancer predisposition disorder, Bloom's syndrome. Our data reveal a mechanism by which cellular responses to stalled replication forks can actively generate genomic alterations and genetic diversity in normal proliferating cells.

Replication stress activates DNA repair synthesis in mitosis.

Oncogene-induced DNA replication stress has been implicated as a driver of tumorigenesis. Many chromosomal rearrangements characteristic of human cancers originate from specific regions of the genome called common fragile sites (CFSs). CFSs are difficult-to-replicate loci that manifest as gaps or breaks on metaphase chromosomes (termed CFS 'expression'), particularly when cells have been exposed to replicative stress. The MUS81-EME1 structure-specific endonuclease promotes the appearance of chromosome gaps or breaks at CFSs following replicative stress. Here we show that entry of cells into mitotic prophase triggers the recruitment of MUS81 to CFSs. The nuclease activity of MUS81 then promotes POLD3-dependent DNA synthesis at CFSs, which serves to minimize chromosome mis-segregation and non-disjunction. We propose that the attempted condensation of incompletely duplicated loci in early mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability (CIN(+)) and replicative stress, we suggest that targeting this pathway could represent a new therapeutic approach.

Tus-Ter as a tool to study site-specific DNA replication perturbation in eukaryotes.

The high-affinity binding of the Tus protein to specific 21-bp sequences, called Ter, causes site-specific, and polar, DNA replication fork arrest in E coli. The Tus-Ter complex serves to coordinate DNA replication with chromosome segregation in this organism. A number of recent and ongoing studies have demonstrated that Tus-Ter can be used as a heterologous tool to generate site-specific perturbation of DNA replication when reconstituted in eukaryotes. Here, we review these recent findings and explore the molecular mechanism by which Tus-Ter mediates replication fork (RF) arrest in the budding yeast, S. cerevisiae. We propose that Tus-Ter is a versatile, genetically tractable, and regulatable RF blocking system that can be utilized for disrupting DNA replication in a diverse range of host cells.

The Escherichia coli Tus-Ter replication fork barrier causes site-specific DNA replication perturbation in yeast.

Replication fork (RF) pausing occurs at both 'programmed' sites and non-physiological barriers (for example, DNA adducts). Programmed RF pausing is required for site-specific DNA replication termination in Escherichia coli, and this process requires the binding of the polar terminator protein, Tus, to specific DNA sequences called Ter. Here, we demonstrate that Tus-Ter modules also induce polar RF pausing when engineered into the Saccharomyces cerevisiae genome. This heterologous RF barrier is distinct from a number of previously characterized, protein-mediated, RF pause sites in yeast, as it is neither Tof1-dependent nor counteracted by the Rrm3 helicase. Although the yeast replisome can overcome RF pausing at Tus-Ter modules, this event triggers site-specific homologous recombination that requires the RecQ helicase, Sgs1, for its timely resolution. We propose that Tus-Ter can be utilized as a versatile, site-specific, heterologous DNA replication-perturbing system, with a variety of potential applications.

How unfinished business from S-phase affects mitosis and beyond.

The eukaryotic cell cycle is conventionally viewed as comprising several discrete steps, each of which must be completed before the next one is initiated. However, emerging evidence suggests that incompletely replicated, or unresolved, chromosomes from S-phase can persist into mitosis, where they present a potential threat to the faithful segregation of sister chromatids. In this review, we provide an overview of the different classes of loci where this 'unfinished S-phase business' can lead to a variety of cytogenetically distinct DNA structures throughout the various steps of mitosis. Furthermore, we discuss the potential ways in which cells might not only tolerate this inevitable aspect of chromosome biology, but also exploit it to assist in the maintenance of genome stability.

MUS81 promotes common fragile site expression.

Fragile sites are chromosomal loci with a propensity to form gaps or breaks during early mitosis, and their instability is implicated as being causative in certain neurological disorders and cancers. Recent work has demonstrated that the so-called common fragile sites (CFSs) often impair the faithful disjunction of sister chromatids in mitosis. However, the mechanisms by which CFSs express their fragility, and the cellular factors required to suppress CFS instability, remain largely undefined. Here, we report that the DNA structure-specific nuclease MUS81-EME1 localizes to CFS loci in early mitotic cells, and promotes the cytological appearance of characteristic gaps or breaks observed at CFSs in metaphase chromosomes. These data indicate that CFS breakage is an active, MUS81-EME1-dependent process, and not a result of inadvertent chromatid rupturing during chromosome condensation. Moreover, CFS cleavage by MUS81-EME1 promotes faithful sister chromatid disjunction. Our findings challenge the prevailing view that CFS breakage is a nonspecific process that is detrimental to cells, and indicate that CFS cleavage actually promotes genome stability.

A novel antirecombinase gains PARIty.

In this issue, Moldovan et al. (2012) report the identification of PARI, a putative human ortholog of the yeast Srs2 protein, which potentially regulates homologous recombination repair via its ability to disrupt the function of RAD51.

Processing of homologous recombination repair intermediates by the Sgs1-Top3-Rmi1 and Mus81-Mms4 complexes.

Homologous recombination repair (HRR) is an evolutionarily conserved cellular process that is important for the maintenance of genome stability during S phase. Inactivation of the Saccharomyces cerevisiae Sgs1-Top3-Rmi1 complex leads to the accumulation of unprocessed, X-shaped HRR intermediates (X structures) following replicative stress. Further characterization of these X structures may reveal why loss of BLM (the human Sgs1 ortholog) leads to the human cancer predisposition disorder, Bloom syndrome. In two recent complementary studies, we examined the nature of the X structures arising in yeast strains lacking Sgs1, Top3 or Rmi1 by identifying which proteins could process these structures in vivo. We revealed that the unprocessed X structures that accumulate in these strains could be resolved by the ectopic overexpression of two different Holliday junction (HJ) resolvases, and that the endogenous Mus81-Mms4 endonuclease could also remove them, albeit slowly. In this review, we discuss the implications of these results and review the putative roles for the Sgs1-Top3-Rmi1 and Mus81-Mms4 complexes in the processing of various types of HRR intermediates during S phase.

Holliday junction-containing DNA structures persist in cells lacking Sgs1 or Top3 following exposure to DNA damage.

The Sgs1-Rmi1-Top3 "dissolvasome" is required for the maintenance of genome stability and has been implicated in the processing of various types of DNA structures arising during DNA replication. Previous investigations have revealed that unprocessed (X-shaped) homologous recombination repair (HRR) intermediates persist when S-phase is perturbed by using methyl methanesulfonate (MMS) in Saccharomyces cerevisiae cells with impaired Sgs1 or Top3. However, the precise nature of these persistent DNA structures remains poorly characterized. Here, we report that ectopic expression of either of two heterologous and structurally unrelated Holliday junction (HJ) resolvases, Escherichia coli RusA or human GEN1(1-527), promotes the removal of these X-structures in vivo. Moreover, other types of DNA replication intermediates, including stalled replication forks and non-HRR-dependent X-structures, are refractory to RusA or GEN1(1-527), demonstrating specificity of these HJ resolvases for MMS-induced X-structures in vivo. These data suggest that the X-structures persisting in cells with impaired Sgs1 or Top3 contain HJs. Furthermore, we demonstrate that Sgs1 directly promotes X-structure removal, because the persistent structures arising in Sgs1-deficient strains are eliminated when Sgs1 is reactivated in vivo. We propose that HJ resolvases and Sgs1-Top3-Rmi1 comprise two independent processes to deal with HJ-containing DNA intermediates arising during HRR in S-phase.

Pathways for Holliday junction processing during homologous recombination in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae Rmi1 protein is a component of the highly conserved Sgs1-Top3-Rmi1 complex. Deletion of SGS1, TOP3, or RMI1 is synthetically lethal when combined with the loss of the Mus81-Mms4 or Slx1-Slx4 endonucleases, which have been implicated in Holliday junction (HJ) resolution. To investigate the causes of this synthetic lethality, we isolated a temperature-sensitive mutant of the RMI1 strain, referred to as the rmi1-1 mutant. At the restrictive temperature, this mutant phenocopies an rmi1Δ strain but behaves like the wild type at the permissive temperature. Following a transient exposure to methyl methanesulfonate, rmi1-1 mutants accumulate unprocessed homologous recombination repair (HRR) intermediates. These intermediates are slowly resolved at the restrictive temperature, revealing a redundant resolution activity when Rmi1 is impaired. This resolution depends on Mus81-Mms4 but not on either Slx1-Slx4 or another HJ resolvase, Yen1. Similar results were also observed when Top3 function was impaired. We propose that the Sgs1-Top3-Rmi1 complex constitutes the main pathway for the processing of HJ-containing HRR intermediates but that Mus81-Mms4 can also resolve these intermediates.

Esc2 and Sgs1 act in functionally distinct branches of the homologous recombination repair pathway in Saccharomyces cerevisiae.

Esc2 is a member of the RENi family of SUMO-like domain proteins and is implicated in gene silencing in Saccharomyces cerevisiae. Here, we identify a dual role for Esc2 during S-phase in mediating both intra-S-phase DNA damage checkpoint signaling and preventing the accumulation of Rad51-dependent homologous recombination repair (HRR) intermediates. These roles are qualitatively similar to those of Sgs1, the yeast ortholog of the human Bloom's syndrome protein, BLM. However, whereas mutation of either ESC2 or SGS1 leads to the accumulation of unprocessed HRR intermediates in the presence of MMS, the accumulation of these structures in esc2 (but not sgs1) mutants is entirely dependent on Mph1, a protein that shows structural similarity to the Fanconi anemia group M protein (FANCM). In the absence of both Esc2 and Sgs1, the intra-S-phase DNA damage checkpoint response is compromised after exposure to MMS, and sgs1esc2 cells attempt to undergo mitosis with unprocessed HRR intermediates. We propose a model whereby Esc2 acts in an Mph1-dependent process, separately from Sgs1, to influence the repair/tolerance of MMS-induced lesions during S-phase.

The RecQ helicase-topoisomerase III-Rmi1 complex: a DNA structure-specific 'dissolvasome'?

RecQ helicases, together with topoisomerase III and Rmi1 family proteins, form an evolutionarily conserved complex that is essential for the maintenance of genome integrity. This complex, which we term RTR, is capable of, or has been implicated in, the processing of a diverse array of DNA structures, and we propose here that it functions in a coordinated fashion as a DNA structure-specific 'dissolvasome'. Little is known about how the RTR complex might be regulated or targeted to various DNA structures in vivo. Recent findings indicate that the components of the RTR complex might activate the cell cycle checkpoint machinery as well as be a target of checkpoint kinases, suggesting that these events are crucial to ensure faithful DNA replication and chromosome segregation.

Shu proteins promote the formation of homologous recombination intermediates that are processed by Sgs1-Rmi1-Top3.

CSM2, PSY3, SHU1, and SHU2 (collectively referred to as the SHU genes) were identified in Saccharomyces cerevisiae as four genes in the same epistasis group that suppress various sgs1 and top3 mutant phenotypes when mutated. Although the SHU genes have been implicated in homologous recombination repair (HRR), their precise role(s) within this pathway remains poorly understood. Here, we have identified a specific role for the Shu proteins in a Rad51/Rad54-dependent HRR pathway(s) to repair MMS-induced lesions during S-phase. We show that, although mutation of RAD51 or RAD54 prevented the formation of MMS-induced HRR intermediates (X-molecules) arising during replication in sgs1 cells, mutation of SHU genes attenuated the level of these structures. Similar findings were also observed in shu1 cells in which Rmi1 or Top3 function was impaired. We propose a model in which the Shu proteins act in HRR to promote the formation of HRR intermediates that are processed by the Sgs1-Rmi1-Top3 complex.

Top3 processes recombination intermediates and modulates checkpoint activity after DNA damage.

Mutation of TOP3 in Saccharomyces cerevisiae causes poor growth, hyperrecombination, and a failure to fully activate DNA damage checkpoints in S phase. Here, we report that overexpression of a dominant-negative allele of TOP3, TOP3(Y356F), which lacks the catalytic (decatenation) activity of Top3, causes impaired S-phase progression and the persistence of abnormal DNA structures (X-shaped DNA molecules) after exposure to methylmethanesulfonate. The impaired S-phase progression is due to a persistent checkpoint-mediated cell cycle delay and can be overridden by addition of caffeine. Hence, the catalytic activity of Top3 is not required for DNA damage checkpoint activation, but it is required for normal S-phase progression after DNA damage. We also present evidence that the checkpoint-mediated cell cycle delay and persistence of X-shaped DNA molecules resulting from overexpression of TOP3(Y356F) are downstream of Rad51 function. We propose that Top3 functions in S phase to both process homologous recombination intermediates and modulate checkpoint activity.

A role for the fission yeast Rqh1 helicase in chromosome segregation.

Schizosaccharomyces pombe Rqh1 protein is a member of the RecQ DNA helicase family. Members of this protein family are mutated in several human genome instability syndromes, including Bloom, Werner and Rothmund-Thomson syndromes. RecQ helicases participate in recombination repair of stalled replication forks or DNA breaks, but the precise mechanisms that lead to the development of cancer in these diseases have remained obscure. Here, we reveal a function for Rqh1 in chromosome segregation even in the absence of exogenous insult to the DNA. We show that cells lacking Rqh1 are delayed in anaphase progression, and show lagging chromosomal DNA, which is particularly apparent in the rDNA locus. This mitotic delay is dependent on the spindle checkpoint, as deletion of mad2 abolishes the delay as well as the accumulation of Cut2 in rqh1delta cells. Furthermore, relieving replication fork arrest in the rDNA repeat by deletion of reb1+ partially suppresses rqh1delta phenotypes. These data are consistent with the function of the Top3-RecQ complex in maintenance of the rDNA structure by processing aberrant chromosome structures arising from DNA replication. The chromosome segregation defects seen in the absence of functional RecQ helicases may contribute to the pathogenesis of human RecQ helicase disorders.

SGS1 is a multicopy suppressor of srs2: functional overlap between DNA helicases.

Sgs1 is a member of the RecQ family of DNA helicases, which have been implicated in genomic stability, cancer and ageing. Srs2 is another DNA helicase that shares several phenotypic features with Sgs1 and double sgs1srs2 mutants have a severe synthetic growth phenotype. This suggests that there may be functional overlap between these two DNA helicases. Consistent with this idea, we found the srs2Delta mutant to have a similar genotoxin sensitivity profile and replicative lifespan to the sgs1Delta mutant. In order to directly test if Sgs1 and Srs2 are functionally interchangeable, the ability of high-copy SGS1 and SRS2 plasmids to complement the srs2Delta and sgs1Delta mutants was assessed. We report here that SGS1 is a multicopy suppressor of the methyl methanesulphonate (MMS) and hydroxyurea sensitivity of the srs2Delta mutant, whereas SRS2 overexpression had no complementing ability in the sgs1Delta mutant. Domains of Sgs1 directly required for processing MMS-induced DNA damage, most notably the helicase domain, are also required for complementation of the srs2Delta mutant. Although SGS1 overexpression was unable to rescue the shortened mean replicative lifespan of the srs2Delta mutant, maximum lifespan was significantly increased by multicopy SGS1. We conclude that Sgs1 is able to partially compensate for the loss of Srs2.