Smc5/6 functions with Sgs1-Top3-Rmi1 to complete chromosome replication at natural pause sites
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ABSTRACT:
PROVIDER: PRJNA614907 | ENA |
REPOSITORIES: ENA
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Project description:Smc5/6 is essential for genome structural integrity by yet unknown mechanisms. Here we find that Smc5/6 co-localizes with the DNA crossed-strand processing complex Sgs1-Top3-Rmi1 (STR) at genomic regions known as natural pausing sites (NPSs) where it facilitates Top3 retention. Individual depletions of STR subunits and Smc5/6 cause similar accumulation of joint molecules (JMs) composed of reversed forks, double Holliday Junctions and hemicatenanes, indicative of Smc5/6 regulating Sgs1 and Top3 DNA processing activities. We isolate an intra-allelic suppressor of smc6-56 proficient in Top3 retention but affected in pathways that act complementarily with Sgs1 and Top3 to resolve JMs arising at replication termination. Upon replication stress, the smc6-56 suppressor requires STR and Mus81-Mms4 functions for recovery, but not Srs2 and Mph1 helicases that prevent maturation of recombination intermediates. Thus, Smc5/6 functions jointly with Top3 and STR to mediate replication completion and influences the function of other DNA crossed-strand processing enzymes at NPSs.
2021-02-03 | GSE147452 | GEO
Project description:Smc5/6 is essential for genome structural integrity by yet unknown mechanisms. Here we find that Smc5/6 co-localizes with the DNA crossed-strand processing complex Sgs1-Top3-Rmi1 (STR) at genomic regions known as natural pausing sites (NPSs) where it facilitates Top3 retention. Individual depletions of STR subunits and Smc5/6 cause similar accumulation of joint molecules (JMs) composed of reversed forks, double Holliday Junctions and hemicatenanes, indicative of Smc5/6 regulating Sgs1 and Top3 DNA processing activities. We isolate an intra-allelic suppressor of smc6-56 proficient in Top3 retention but affected in pathways that act complementarily with Sgs1 and Top3 to resolve JMs arising at replication termination. Upon replication stress, the smc6-56 suppressor requires STR and Mus81-Mms4 functions for recovery, but not Srs2 and Mph1 helicases that prevent maturation of recombination intermediates. Thus, Smc5/6 functions jointly with Top3 and STR to mediate replication completion and influences the function of other DNA crossed-strand processing enzymes at NPSs.
| S-EPMC8032827 | 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.
2015-11-19 | GSE72241 | GEO
Project description:Replication forks face multiple obstacles that slow their progression. By two-dimensional gel analysis, yeast forks pause at stable DNA protein complexes, and this pausing is greatly increased in the absence of the Rrm3 helicase. We used a genome wide approach to identify 96 sites of very high DNA polymerase binding in wild type cells. Most of these binding sites were not previously identified pause sites. Rather, the most highly represented genomic category among high DNA polymerase binding sites was the open reading frames (ORFs) of highly transcribed RNA polymerase II genes. Twice as many pause sites were identified in rrm3 compared to wild type cells as pausing in this strain occurred at both highly transcribed RNA polymerase II genes and the previously identified protein DNA complexes. ORFs of highly transcribed RNA polymerase II genes are the first class of natural pause sites that are not exacerbated in rrm3 cells. We alse mapped pause sites using a second replication fork component, Rrm3-13MYC and got similar results. Genomic input (labelled with Cy3) and IP'ed DNA (labelled with Cy5) using a MYC Ab of either DNA Pol2-13MYC or Rrm3-MYC from asynchronously grown S. cerevisiae cells in rich media were hybridized to whole-genome PCR-based arrays containing ORF and intergenic regions of the entire genome (Ivery et al 2001). At least three biological replication and one technical replicate (dye swap) were performed. Log2 transformed median normalized ratios (IP/IN) were averaged for each experiment and significant peaks of either DNA Pol2 or Rrm3 association were identified
2009-06-15 | E-GEOD-16218 | biostudies-arrayexpress
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. BrdU incorporation profiles by ssDNA-BrdU IP on chip have been generated as described (Katou et al., 2003). Protein binding profiles by ChIP-chip analysis were generated as described (Bermejo et al., 2009). Labeled probes were hybridized to Affymetrix S.cerevisiae Tiling 1.0 (P/N 900645) arrays and processed with TAS software.
2015-11-19 | E-GEOD-72241 | biostudies-arrayexpress
Project description:Replication forks face multiple obstacles that slow their progression. By two-dimensional gel analysis, yeast forks pause at stable DNA protein complexes, and this pausing is greatly increased in the absence of the Rrm3 helicase. We used a genome wide approach to identify 96 sites of very high DNA polymerase binding in wild type cells. Most of these binding sites were not previously identified pause sites. Rather, the most highly represented genomic category among high DNA polymerase binding sites was the open reading frames (ORFs) of highly transcribed RNA polymerase II genes. Twice as many pause sites were identified in rrm3 compared to wild type cells as pausing in this strain occurred at both highly transcribed RNA polymerase II genes and the previously identified protein DNA complexes. ORFs of highly transcribed RNA polymerase II genes are the first class of natural pause sites that are not exacerbated in rrm3 cells. We alse mapped pause sites using a second replication fork component, Rrm3-13MYC and got similar results.
2009-06-15 | GSE16218 | GEO
Project description:During meiosis, Structural Maintenance of Chromosome (SMC) complexes underpin two fundamental features of meiosis: homologous recombination and chromosome segregation. While meiotic functions of the cohesin and condensin complexes have been delineated, the role of the third SMC complex, Smc5/6, remains enigmatic. Diminished Smc5/6 function causes severe defects in nuclear division, but the underlying causes of these defects remain unclear. Here we identify specific, essential meiotic functions for the Smc5/6 complex in homologous recombination and regulation of cohesin. We show that Smc5/6 is enriched at centromeres and cohesin-association sites where it regulates sister-chromatid cohesion and the timely removal of cohesin from chromosomal arms, respectively. Smc5/6 also localizes to recombination hotspots, where it promotes normal formation and resolution of joint-molecule intermediates. Furthermore, we find that Smc5/6 specifically promotes resolution of joint molecules via the XPF-family endonuclease, Mus81-Mms4Eme1. We propose that Smc5/6 acts as a chaperone for M-bM-^@M-^XmitoticM-bM-^@M-^Y-like recombination processes during meiosis. ChIP-chip was used to compare Smc5 localization in wild-type and spo11 strains
2013-12-27 | E-GEOD-44852 | biostudies-arrayexpress
Project description:During meiosis, Structural Maintenance of Chromosome (SMC) complexes underpin two fundamental features of meiosis: homologous recombination and chromosome segregation. While meiotic functions of the cohesin and condensin complexes have been delineated, the role of the third SMC complex, Smc5/6, remains enigmatic. Diminished Smc5/6 function causes severe defects in nuclear division, but the underlying causes of these defects remain unclear. Here we identify specific, essential meiotic functions for the Smc5/6 complex in homologous recombination and regulation of cohesin. We show that Smc5/6 is enriched at centromeres and cohesin-association sites where it regulates sister-chromatid cohesion and the timely removal of cohesin from chromosomal arms, respectively. Smc5/6 also localizes to recombination hotspots, where it promotes normal formation and resolution of joint-molecule intermediates. Furthermore, we find that Smc5/6 specifically promotes resolution of joint molecules via the XPF-family endonuclease, Mus81-Mms4Eme1. We propose that Smc5/6 acts as a chaperone for ‘mitotic’-like recombination processes during meiosis.
2013-12-27 | GSE44852 | GEO
Project description:The human SMC5-SMC6 complex (SMC5/6) is a conserved guardian of genome stability and an emerging component of the antiviral response in animals and plants. In yeast, Smc5/6 is recruited to and loaded on chromatin by the poorly conserved Nse5-Nse6 heterodimer (Nse5/6), which remains largely undefined in humans. Here we report that SIMC1 is an Nse5 orthologue and novel subunit of the human SMC5/6 complex. SIMC1 binds to SLF2, the proposed Nse6 orthologue, forming a stable complex structurally similar to yeast Nse5/6. SIMC1 localizes SMC5/6 to polyomavirus replication centers in a SUMO interaction-dependent manner. Further structure prediction analyses indicate that SLF1, a putative Nse5 counterpart that recruits SMC5/6 to DNA lesions, interacts with SLF2 in a very similar manner as SIMC1. Consistently, SIMC1 and SLF1 form mutually exclusive complexes with SLF2. Thus, both SIMC1 and SLF1 act as orthologues of yeast Nse5 to independently direct the functions of SMC5/6.
2022-11-29 | PXD033923 | Pride
Project description:Beyond its essential roles in ensuring faithful chromosome segregation and genomic stability, the human Smc5/6 complex acts as an antiviral factor. It binds to and impedes the transcription of extrachromosomal DNA templates; an ability which is lost upon chromosomal DNA integration. How the complex distinguishes among different DNA templates is unknown. Here we show that, in human cells, Smc5/6 preferentially binds to circular rather than linear extrachromosomal DNA. We further show that this binding is unlikely due to differences in the chromatin composition. Instead, the transcriptional process, per se, and more specifically the accumulation of DNA secondary structures known to be substrates for topoisomerases, is responsible for Smc5/6 recruitment. Those findings, in conjunction with our genome-wide Smc5/6 binding analysis showing that Smc5/6 localises at few but highly transcribe chromosome loci, reveal a previously unforeseen role of Smc5/6 in DNA topology management during transcription.
2024-06-10 | GSE231328 | GEO