The Smc5/6 complex and Esc2 influence multiple replication-associated recombination processes in Saccharomyces cerevisiae.
ABSTRACT: Replication-associated recombinational repair is important for genome duplication and cell survival under DNA damage conditions. Several nonclassical recombination factors have been implicated in this process, but their functional relationships are not clear. Here, we show that three of these factors, Mph1, Mms2, and the Shu complex, can act independently to promote the formation of recombination intermediates during impaired replication. However, their functions become detrimental when cells lack the Smc5/6 complex or Esc2. We show that mph1Delta, mms2Delta, and shu1Delta suppress the sensitivity to the replication-blocking agent methylmethane sulfonate (MMS) in smc6 mutants, with double deletions conferring stronger suppression. These deletion mutations also rescue the MMS sensitivity of esc2Delta cells. In addition, two-dimensional gel analysis demonstrates that mph1Delta, mms2Delta, and shu1Delta each reduce the level of recombination intermediates in an smc6 mutant when cells replicate in the presence of MMS, and that double deletions lead to a greater reduction. Our work thus suggests that Mph1, Mms2, and the Shu complex can function in distinct pathways in replication-associated recombinational repair and that the Smc5/6 complex and Esc2 prevent the accumulation of toxic recombination intermediates generated in these processes.
Project description:DNA damage checkpoint and recombinational repair are both important for cell survival of replication stress. Because these two processes influence each other, isolation of their respective contributions is challenging. Research in budding yeast shows that removal of the DNA helicase Mph1 improves survival of cells with defective Smc5/6 complex under replication stress. mph1 is known to reduce the levels of recombination intermediates in smc6 mutants. Here, we show that mph1 also hyperactivates the Mec1 checkpoint. We dissect the effects of recombination regulation and checkpoint hyperactivation by altering the checkpoint circuitry to enhance checkpoint signaling without reducing recombination intermediate levels. We show that these approaches, similar to mph1, lead to better survival of smc6 cells upon transient replication stress, likely by ameliorating replication and chromosomal segregation defects. Unlike mph1, however, they do not suppress smc6 sensitivity to chronic stress. Conversely, reducing the checkpoint response does not impair survival of smc6 mph1 mutants under chronic stress. These results suggest a two-phase model in which smc6 mutant survival upon transient replication stress can be improved by enhancing Mec1 checkpoint signaling, whereas smc6 sensitivity to chronic stress can be overcome by reducing recombination intermediates.
Project description: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.
Project description:The essential and evolutionarily conserved Smc5-Smc6 complex (Smc5/6) is critical for the maintenance of genome stability. Partial loss of Smc5/6 function yields several defects in DNA repair, which are rescued by inactivation of the homologous recombination (HR) machinery. Thus HR is thought to be toxic to cells with defective Smc5/6. Recent work has highlighted a role for Smc5/6 and the Sgs1 DNA helicase in preventing the accumulation of unresolved HR intermediates. Here we investigate how deletion of MPH1, encoding the orthologue of the human FANCM DNA helicase, rescues the DNA damage sensitivity of smc5/6 but not sgs1? mutants. We find that MPH1 deletion diminishes accumulation of HR intermediates within both smc5/6 and sgs1? cells, suggesting that MPH1 deletion is sufficient to decrease the use of template switch recombination (TSR) to bypass DNA lesions. We further explain how avoidance of TSR is nonetheless insufficient to rescue defects in sgs1? mutants, by demonstrating a requirement for Sgs1, along with the post-replicative repair (PRR) and HR machinery, in a pathway that operates in mph1? mutants. In addition, we map the region of Mph1 that binds Smc5, and describe a novel allele of MPH1 encoding a protein unable to bind Smc5 (mph1-?60). Remarkably, mph1-?60 supports normal growth and responses to DNA damaging agents, indicating that Smc5/6 does not simply restrain the recombinogenic activity of Mph1 via direct binding. These data as a whole highlight a role for Smc5/6 and Sgs1 in the resolution of Mph1-dependent HR intermediates.
Project description:Conserved, multitasking DNA helicases mediate diverse DNA transactions and are relevant for human disease pathogenesis. These helicases and their regulation help maintain genome stability during DNA replication and repair. We show that the structural maintenance of chromosome complex Smc5-Smc6 restrains the replication fork regression activity of Mph1 helicase, but not its D loop disruptive activity. This regulatory mechanism enables flexibility in replication fork repair without interfering with DNA break repair. In vitro studies find that Smc5-Smc6 binds to a Mph1 region required for efficient fork regression, preventing assembly of Mph1 oligomers at the junction of DNA forks. In vivo impairment of this regulatory mechanism compensates for the inactivation of another fork regression helicase and increases reliance on joint DNA structure removal or avoidance. Our findings provide molecular insights into replication fork repair regulation and uncover a role of Smc5-Smc6 in directing Mph1 activity toward a specific biochemical outcome.
Project description:The evolutionarily conserved Smc5/6 complex is implicated in recombinational repair, but its function in this process has been elusive. Here we report that the budding yeast Smc5/6 complex directly binds to the DNA helicase Mph1. Mph1 and its helicase activity define a replication-associated recombination subpathway. We show that this pathway is toxic when the Smc5/6 complex is defective, because mph1Delta and its helicase mutations suppress multiple defects in mutants of the Smc5/6 complex, including their sensitivity to replication-blocking agents, growth defects, and inefficient chromatid separation, whereas MPH1 overexpression exacerbates some of these defects. We further demonstrate that Mph1 and its helicase activity are largely responsible for the accumulation of potentially deleterious recombination intermediates in mutants of the Smc5/6 complex. We also present evidence that mph1Delta does not alleviate sensitivity to DNA damage or the accumulation of recombination intermediates in cells lacking Sgs1, which is thought to function together with the Smc5/6 complex. Thus, our results reveal a function of the Smc5/6 complex in the Mph1-dependent recombinational subpathway that is distinct from Sgs1. We suggest that the Smc5/6 complex can counteract/modulate a pro-recombinogenic function of Mph1 or facilitate the resolution of recombination structures generated by Mph1.
Project description:Meiosis, a specialized cell division with a single cycle of DNA replication round and two consecutive rounds of nuclear segregation, allows for the exchange of genetic material between parental chromosomes and the formation of haploid gametes. The structural maintenance of chromosome (SMC) proteins aid manipulation of chromosome structures inside cells. Eukaryotic SMC complexes include cohesin, condensin and the Smc5-Smc6 complex. Meiotic roles have been discovered for cohesin and condensin. However, although Smc5-Smc6 is known to be required for successful meiotic divisions, the meiotic functions of the complex are not well understood. Here we show that the Smc5-Smc6 complex localizes to specific chromosome regions during meiotic prophase I. We report that meiotic cells lacking Smc5-Smc6 undergo catastrophic meiotic divisions as a consequence of unresolved linkages between chromosomes. Surprisingly, meiotic segregation defects are not rescued by abrogation of Spo11-induced meiotic recombination, indicating that at least some chromosome linkages in smc5-smc6 mutants originate from other cellular processes. These results demonstrate that, as in mitosis, Smc5-Smc6 is required to ensure proper chromosome segregation during meiosis by preventing aberrant recombination intermediates between homologous chromosomes.
Project description:Telomeres are repetitive nucleoprotein structures that cap the ends of chromosomes. Without telomerase, telomeres shorten with replication and eventually signal cell cycle arrest (cell senescence). Homologous recombination (HR)-based mechanisms slow senescence, and distinct HR mechanisms support the growth of the rare survivors of senescence. Here, we report novel roles for the post-translational modification of small ubiquitin-like modifier (SUMO) in regulating the rate of senescence in Saccharomyces cerevisiae telomerase mutants. We identify Mms21 as the relevant SUMO E3 ligase and demonstrate that cells lacking Mms21-dependent sumoylation accumulate HR intermediates selectively at telomeres during senescence. One target of Mms21-dependent sumoylation is the cohesin- and condensin-related Smc5-Smc6 complex (Smc5/6). We show that hypomorphic smc5 or smc6 alleles exhibit phenotypes similar to mms21 sumoylation-deficient mutants with regard to senescence and the accumulation of unresolved HR intermediates. Further, we provide evidence that Mms21 and Smc5/6 prevent aberrant recombination between sister telomeres and also globally facilitate resolution of sister chromatid HR intermediates.
Project description:Smc5 and Smc6 proteins form a heterodimeric SMC (structural maintenance of chromosome) protein complex like SMC1-SMC3 cohesin and SMC2-SMC4 condensin, and they associate with non-SMC proteins Nse1 and Nse2 stably and Rad60 transiently. This multiprotein complex plays an essential role in maintaining chromosome integrity and repairing DNA double strand breaks (DSBs). This study characterizes a Schizosaccharomyces pombe mutant rad62-1, which is hypersensitive to methyl methanesulfonate (MMS) and synthetically lethal with rad2 (a feature of recombination mutants). rad62-1 is hypersensitive to UV and gamma rays, epistatic with rhp51, and defective in repair of DSBs. rad62 is essential for viability and genetically interacts with rad60, smc6, and brc1. Rad62 protein physically associates with the Smc5-6 complex. rad62-1 is synthetically lethal with mutations in the genes promoting recovery from stalled replication, such as rqh1, srs2, and mus81, and those involved in nucleotide excision repair like rad13 and rad16. These results suggest that Rad62, like Rad60, in conjunction with the Smc5-6 complex, plays an essential role in maintaining chromosome integrity and recovery from stalled replication by recombination.
Project description:Recombination is important for DNA repair, but it can also contribute to genome rearrangements. RecQ helicases, including yeast Sgs1 and human BLM, safeguard genome integrity through their functions in DNA recombination. Sgs1 prevents the accumulation of Rad51-dependent sister chromatid junctions at damaged replication forks, and its functionality seems to be regulated by Ubc9- and Mms21-dependent sumoylation. We show that mutations in Smc5-6 and Esc2 also lead to an accumulation of recombinogenic structures at damaged replication forks. Because Smc5-6 is sumoylated in an Mms21-dependent manner, this finding suggests that Smc5-6 may be a crucial target of Mms21 implicated in this process. Our data reveal that Smc5-6 and Esc2 are required to tolerate DNA damage and that their functionality is critical in genotoxic conditions in the absence of Sgs1. As reported previously for Sgs1 and Smc5-6, we find that Esc2 physically interacts with Ubc9 and SUMO. This interaction is correlated with the ability of Esc2 to promote DNA damage tolerance. Collectively, these data suggest that Esc2 and Smc5-6 act in concert with Sgs1 to prevent the accumulation of recombinogenic structures at damaged replication forks, likely by integrating sumoylation activities to regulate the repair pathways in response to damaged DNA.
Project description:The Smc5/6 structural maintenance of chromosomes complex is required for efficient homologous recombination (HR). Defects in Smc5/6 result in chromosome mis-segregation and fragmentation. By characterising two Schizosaccharomyces pombe smc6 mutants, we define two separate functions for Smc5/6 in HR. The first represents the previously described defect in processing recombination-dependent DNA intermediates when replication forks collapse, which leads to increased rDNA recombination. The second novel function defines Smc5/6 as a positive regulator of recombination in the rDNA and correlates mechanistically with a requirement to load RPA and Rad52 onto chromatin genome-wide when replication forks are stably stalled by nucleotide depletion. Rad52 is required for all HR repair, but Rad52 loading in response to replication fork stalling is unexpected and does not correlate with damage-induced foci. We propose that Smc5/6 is required to maintain stalled forks in a stable recombination-competent conformation primed for replication restart.