The Smc5/6 complex regulates the yeast Mph1 helicase at RNA-DNA hybrid-mediated DNA damage.
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ABSTRACT:
SUBMITTER: Lafuente-Barquero J
PROVIDER: S-EPMC5760084 | biostudies-literature | 2017 Dec
REPOSITORIES: biostudies-literature
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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.
| S-EPMC2795505 | biostudies-literature
Project description:The detection of DNA damage activates DNA repair pathways and checkpoints to allow time for repair. Ultimately, these responses must be coordinated to ensure that cell cycle progression is halted until repair is completed. Several multiprotein complexes containing members of the structural maintenance of chromosomes family of proteins have been described, including the condensin and cohesin complexes, that are critical for chromosomal organization. Here we show that the Smc5/Smc6 (Smc5/6) complex is required for a coordinated response to DNA damage and normal chromosome integrity. Fission yeast cells lacking functional Smc6 initiate a normal checkpoint response to DNA damage, culminating in the phosphorylation and activation of the Chk1 protein kinase. Despite this, cells enter a lethal mitosis, presumably without completion of DNA repair. Another subunit of the complex, Nse1, is a conserved member of this complex and is also required for this response. We propose that the failure to maintain a checkpoint response stems from the lack of ongoing DNA repair or from defective chromosomal organization, which is the signal to maintain a checkpoint arrest. The Smc5/6 complex is fundamental to genome integrity and may function with the condensin and cohesin complexes in a coordinated manner.
| S-EPMC343814 | biostudies-literature
Project description:The Saccharomyces cerevisiae Fkh1 protein has roles in cell-cycle regulated transcription as well as a transcription-independent role in recombination donor preference during mating-type switching. The conserved FHA domain of Fkh1 regulates donor preference by juxtaposing two distant regions on chromosome III to promote their recombination. A model posits that this Fkh1-mediated long-range chromosomal juxtaposition requires an interaction between the FHA domain and a partner protein(s), but to date no relevant partner has been described. In this study, we used structural modeling, 2-hybrid assays, and mutational analyses to show that the predicted phosphothreonine-binding FHA domain of Fkh1 interacted with multiple partner proteins. The Fkh1 FHA domain was important for its role in cell-cycle regulation, but no single interaction partner could account for this role. In contrast, Fkh1's interaction with the Mph1 DNA repair helicase regulated donor preference during mating-type switching. Using 2-hybrid assays, co-immunoprecipitation, and fluorescence anisotropy, we mapped a discrete peptide within the regulatory Mph1 C-terminus required for this interaction and identified two threonines that were particularly important. In vitro binding experiments indicated that at least one of these threonines had to be phosphorylated for efficient Fkh1 binding. Substitution of these two threonines with alanines (mph1-2TA) specifically abolished the Fkh1-Mph1 interaction in vivo and altered donor preference during mating-type switching to the same degree as mph1?. Notably, the mph1-2TA allele maintained other functions of Mph1 in genome stability. Deletion of a second Fkh1-interacting protein encoded by YMR144W also resulted in a change in Fkh1-FHA-dependent donor preference. We have named this gene FDO1 for Forkhead one interacting protein involved in donor preference. We conclude that a phosphothreonine-mediated protein-protein interface between Fkh1-FHA and Mph1 contributes to a specific long-range chromosomal interaction required for mating-type switching, but that Fkh1-FHA must also interact with several other proteins to achieve full functionality in this process.
| S-EPMC4892509 | biostudies-literature
Project description:Double strand breaks (DSBs) can be repaired via either Non-Homologous End Joining (NHEJ) or Homology directed Repair (HR). Telomeres, which resemble DSBs, are refractory to repair events in order to prevent chromosome end fusions and genomic instability. In some rare instances telomeres engage in Break-Induced Replication (BIR), a type of HR, in order to maintain telomere length in the absence of the enzyme telomerase. Here we have investigated how the yeast helicase, Mph1, affects DNA repair at both DSBs and telomeres. We have found that overexpressed Mph1 strongly inhibits BIR at internal DSBs however allows it to proceed at telomeres. Furthermore, while overexpressed Mph1 potently inhibits NHEJ at telomeres it has no effect on NHEJ at DSBs within the chromosome. At telomeres Mph1 is able to promote telomere uncapping and the accumulation of ssDNA, which results in premature senescence in the absence of telomerase. We propose that Mph1 is able to direct repair towards HR (thereby inhibiting NHEJ) at telomeres by remodeling them into a nuclease-sensitive structure, which promotes the accumulation of a recombinogenic ssDNA intermediate. We thus put forward that Mph1 is a double-edge sword at the telomere, it prevents NHEJ, but promotes senescence in cells with dysfunctional telomeres by increasing the levels of ssDNA.
| S-EPMC3407055 | biostudies-literature
Project description:Double-strand breaks (DSBs) are the most lethal form of DNA damage. Transcriptional activity at DSBs, as well as transcriptional repression around DSBs, are both required for efficient DNA repair. The chromatin landscape defines and coordinates these two opposing events. However, how the open and condensed chromatin architecture is regulated remains unclear. Here, we show that the GATAD2B-NuRD complex associates with DSBs in a transcription- and DNA:RNA hybrid-dependent manner, to promote histone deacetylation and chromatin condensation. This activity establishes a spatio-temporal boundary between open and closed chromatin, which is necessary for the correct termination of DNA end resection. The lack of the GATAD2B-NuRD complex leads to chromatin hyper-relaxation and extended DNA end resection, resulting in HR repair failure. Our results suggest that the GATAD2B-NuRD complex is a key coordinator of the dynamic interplay between transcription and the chromatin landscape, underscoring its biological significance in the RNA-dependent DNA damage response.
| S-SCDT-10_1038-S44318-024-00111-7 | biostudies-other
Project description:The family of RecQ helicases is evolutionary conserved from bacteria to humans and play key roles in genome stability. The budding yeast RecQ helicase Sgs1 has been implicated in several key processes during the repair of DNA damage by homologous recombination as part of the STR complex (Sgs1-Top3-Rmi1). Limited information on how is Sgs1 recruited and regulated at sites of damage is available. Recently, we and others have uncover a direct link between the Smc5/6 complex and Sgs1. Most roles of Sgs1 during recombination, including DNA end resection, Holiday junction dissolution, and crossover suppression, are regulated through Mms21-dependent SUMOylation. Smc5/6 first acts as a recruiting platform for STR and then SUMOylates STR components to regulate their function. Importantly, the assembly of STR is totally independent of Smc5/6. Here, we provide a brief overview of STR regulation by Smc5/6.
| S-EPMC5422486 | biostudies-literature
Project description:RECQ5 DNA helicase suppresses homologous recombination (HR) possibly through disruption of RAD51 filaments. Here, we show that RECQ5 is constitutively associated with the MRE11-RAD50-NBS1 (MRN) complex, a primary sensor of DNA double-strand breaks (DSBs) that promotes DSB repair and regulates DNA damage signaling via activation of the ATM kinase. Experiments with purified proteins indicated that RECQ5 interacts with the MRN complex through both MRE11 and NBS1. Functional assays revealed that RECQ5 specifically inhibited the 3'-->5' exonuclease activity of MRE11, while MRN had no effect on the helicase activity of RECQ5. At the cellular level, we observed that the MRN complex was required for the recruitment of RECQ5 to sites of DNA damage. Accumulation of RECQ5 at DSBs was neither dependent on MDC1 that mediates binding of MRN to DSB-flanking chromatin nor on CtIP that acts in conjunction with MRN to promote resection of DSBs for repair by HR. Collectively, these data suggest that the MRN complex recruits RECQ5 to sites of DNA damage to regulate DNA repair.
| S-EPMC2677886 | biostudies-literature
Project description:Saccharomyces cerevisiae Mph1 is a 3-5' DNA helicase, required for the maintenance of genome integrity. In order to understand the ATPase/helicase role of Mph1 in genome stability, we characterized its helicase activity with a variety of DNA substrates, focusing on its action on junction structures containing three or four DNA strands. Consistent with its 3' to 5' directionality, Mph1 displaced 3'-flap substrates in double-fixed or equilibrating flap substrates. Surprisingly, Mph1 displaced the 5'-flap strand more efficiently than the 3' flap strand from double-flap substrates, which is not expected for a 3-5' DNA helicase. For this to occur, Mph1 required a threshold size (>5 nt) of 5' single-stranded DNA flap. Based on the unique substrate requirements of Mph1 defined in this study, we propose that the helicase/ATPase activity of Mph1 play roles in converting multiple-stranded DNA structures into structures cleavable by processing enzymes such as Fen1. We also found that the helicase activity of Mph1 was used to cause structural alterations required for restoration of replication forks stalled due to damaged template. The helicase properties of Mph1 reported here could explain how it resolves D-loop structure, and are in keeping with a model proposed for the error-free damage avoidance pathway.
| S-EPMC3300029 | biostudies-literature
Project description:The essential functions of the conserved Smc5/6 complex remain elusive. To uncover its roles in genome maintenance, we established Saccharomyces cerevisiae cell-cycle-regulated alleles that enable restriction of Smc5/6 components to S or G2/M. Unexpectedly, the essential functions of Smc5/6 segregated fully and selectively to G2/M. Genetic screens that became possible with generated alleles identified processes that crucially rely on Smc5/6 specifically in G2/M: metabolism of DNA recombination structures triggered by endogenous replication stress, and replication through natural pausing sites located in late-replicating regions. In the first process, Smc5/6 modulates remodeling of recombination intermediates, cooperating with dissolution activities. In the second, Smc5/6 prevents chromosome fragility and toxic recombination instigated by prolonged pausing and the fork protection complex, Tof1-Csm3. Our results thus dissect Smc5/6 essential roles and reveal that combined defects in DNA damage tolerance and pausing site-replication cause recombination-mediated DNA lesions, which we propose to drive developmental and cancer-prone disorders.
| S-EPMC4691243 | biostudies-literature
Project description:The structural maintenance of chromosomes complex SMC5/6 is thought to be essential for DNA repair and chromosome segregation during mitosis and meiosis. To determine the requirements of the SMC5/6 complex during mouse spermatogenesis we combined a conditional knockout allele for Smc5, with four germ cell-specific Cre-recombinase transgenes, Ddx4-Cre, Stra8-Cre, Spo11-Cre, and Hspa2-Cre, to mutate Smc5 in spermatogonia, in spermatocytes before meiotic entry, during early meiotic stages, and during midmeiotic stages, respectively. Conditional mutation of Smc5 resulted in destabilization of the SMC5/6 complex. Despite this, we observed only mild defects in spermatogenesis. Mutation of Smc5 mediated by Ddx4-Cre and Stra8-Cre resulted in partial loss of preleptotene spermatocytes; however, spermatogenesis progresses and mice are fertile. Mutation of Smc5 via Spo11-Cre or Hspa2-Cre did not result in detectable defects of spermatogenesis. Upon exposure to gamma irradiation or etoposide treatment, each conditional Smc5 mutant demonstrated an increase in the number of enlarged round spermatids with multiple acrosomes and supernumerary chromosome content. We propose that the SMC5/6 complex is not acutely required for premeiotic DNA replication and meiotic progression during mouse spermatogenesis; however, when germ cells are challenged by exogenous DNA damage, the SMC5/6 complex ensures genome integrity, and thus, fertility.
| S-EPMC6333175 | biostudies-literature