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Cohesin Causes Replicative DNA Damage by Trapping DNA Topological Stress.

ABSTRACT: DNA topological stress inhibits DNA replication fork (RF) progression and contributes to DNA replication stress. In Saccharomyces cerevisiae, we demonstrate that centromeric DNA and the rDNA array are especially vulnerable to DNA topological stress during replication. The activity of the SMC complexes cohesin and condensin are linked to both the generation and repair of DNA topological-stress-linked damage in these regions. At cohesin-enriched centromeres, cohesin activity causes the accumulation of DNA damage, RF rotation, and pre-catenation, confirming that cohesin-dependent DNA topological stress impacts on normal replication progression. In contrast, at the rDNA, cohesin and condensin activity inhibit the repair of damage caused by DNA topological stress. We propose that, as well as generally acting to ensure faithful genetic inheritance, SMCs can disrupt genome stability by trapping DNA topological stress.

SUBMITTER: Minchell NE

PROVIDER: S-EPMC7242899 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Cohesin Causes Replicative DNA Damage by Trapping DNA Topological Stress.

Minchell Nicola Elizabeth NE Keszthelyi Andrea A Baxter Jonathan J

Molecular cell 20200406 4

DNA topological stress inhibits DNA replication fork (RF) progression and contributes to DNA replication stress. In Saccharomyces cerevisiae, we demonstrate that centromeric DNA and the rDNA array are especially vulnerable to DNA topological stress during replication. The activity of the SMC complexes cohesin and condensin are linked to both the generation and repair of DNA topological-stress-linked damage in these regions. At cohesin-enriched centromeres, cohesin activity causes the accumulatio ...[more]

PMID: 32259483

Similar Datasets

Cohesin causes replicative DNA damage by trapping DNA topological stress

Project description:DNA topological stress inhibits DNA replication fork (RF) progression and contributes to DNA replication stress. In Saccharomyces cerevisiae we demonstrate that centromeric DNA and the rDNA array are especially vulnerable to DNA topological stress during replication. The activity of the SMC complexes cohesin and condensin are linked to both the generation and repair of DNA topological stress linked damage in these regions. At cohesin enriched centromeres cohesin activity causes the accumulation of DNA damage, RF rotation and precatenation, confirming that cohesin dependent DNA topological stress impacts on normal replication progression. In contrast, at the rDNA cohesin and condensin activity inhibit the repair of damage caused by DNA topological stress. We propose that as well as generally acting to ensure faithful genetic inheritance, SMCs can disrupt genome stability by trapping DNA topological stress.

2020-03-03 | GSE131558 | GEO

Cohesin causes replicative DNA damage by trapping DNA topological stress

Project description:Cohesin causes replicative DNA damage by trapping DNA topological stress

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Proapoptotic Bid mediates the Atr-directed DNA damage response to replicative stress.

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Biochemical reconstitution of topological DNA binding by the cohesin ring.

Project description:Cohesion between sister chromatids, mediated by the chromosomal cohesin complex, is a prerequisite for faithful chromosome segregation in mitosis. Cohesin also has vital roles in DNA repair and transcriptional regulation. The ring-shaped cohesin complex is thought to encircle sister DNA strands, but its molecular mechanism of action is poorly understood and the biochemical reconstitution of cohesin activity in vitro has remained an unattained goal. Here we reconstitute cohesin loading onto DNA using purified fission yeast cohesin and its loader complex, Mis4(Scc2)-Ssl3(Scc4) (Schizosaccharomyces pombe gene names appear throughout with their more commonly known Saccharomyces cerevisiae counterparts added in superscript). Incubation of cohesin with DNA leads to spontaneous topological loading, but this remains inefficient. The loader contacts cohesin at multiple sites around the ring circumference, including the hitherto enigmatic Psc3(Scc3) subunit, and stimulates cohesin's ATPase, resulting in efficient topological loading. The in vitro reconstitution of cohesin loading onto DNA provides mechanistic insight into the initial steps of the establishment of sister chromatid cohesion and other chromosomal processes mediated by cohesin.

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Unscheduled expression of CDC25B in S-phase leads to replicative stress and DNA damage.

Project description:BackgroundCDC25B phosphatase is a cell cycle regulator that plays a critical role in checkpoint control. Up-regulation of CDC25B expression has been documented in a variety of human cancers, however, the relationships with the alteration of the molecular mechanisms that lead to oncogenesis still remain unclear. To address this issue we have investigated, in model cell lines, the consequences of unscheduled and elevated CDC25B levels.ResultsWe report that increased CDC25B expression leads to DNA damage in the absence of genotoxic treatment. H2AX phosphorylation is detected in S-phase cells and requires active replication. We also report that CDC25B expression impairs DNA replication and results in an increased recruitment of the CDC45 replication factor onto chromatin. Finally, we observed chromosomal aberrations that are also enhanced upon CDC25B expression.ConclusionOverall, our results demonstrate that a moderate and unscheduled increase in CDC25B level, as observed in a number of human tumours, is sufficient to overcome the S-phase checkpoint efficiency thus leading to replicative stress and genomic instability.

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Polymerase δ deficiency causes syndromic immunodeficiency with replicative stress.

Project description:Polymerase δ is essential for eukaryotic genome duplication and synthesizes DNA at both the leading and lagging strands. The polymerase δ complex is a heterotetramer comprising the catalytic subunit POLD1 and the accessory subunits POLD2, POLD3, and POLD4. Beyond DNA replication, the polymerase δ complex has emerged as a central element in genome maintenance. The essentiality of polymerase δ has constrained the generation of polymerase δ-knockout cell lines or model organisms and, therefore, the understanding of the complexity of its activity and the function of its accessory subunits. To our knowledge, no germline biallelic mutations affecting this complex have been reported in humans. In patients from 2 independent pedigrees, we have identified what we believe to be a novel syndrome with reduced functionality of the polymerase δ complex caused by germline biallelic mutations in POLD1 or POLD2 as the underlying etiology of a previously unknown autosomal-recessive syndrome that combines replicative stress, neurodevelopmental abnormalities, and immunodeficiency. Patients' cells showed impaired cell-cycle progression and replication-associated DNA lesions that were reversible upon overexpression of polymerase δ. The mutations affected the stability and interactions within the polymerase δ complex or its intrinsic polymerase activity. We believe our discovery of human polymerase δ deficiency identifies the central role of this complex in the prevention of replication-related DNA lesions, with particular relevance to adaptive immunity.

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The Cohesin Ring Uses Its Hinge to Organize DNA Using Non-topological as well as Topological Mechanisms.

Project description:As predicted by the notion that sister chromatid cohesion is mediated by entrapment of sister DNAs inside cohesin rings, there is perfect correlation between co-entrapment of circular minichromosomes and sister chromatid cohesion. In most cells where cohesin loads without conferring cohesion, it does so by entrapment of individual DNAs. However, cohesin with a hinge domain whose positively charged lumen is neutralized loads and moves along chromatin despite failing to entrap DNAs. Thus, cohesin engages chromatin in non-topological, as well as topological, manners. Since hinge mutations, but not Smc-kleisin fusions, abolish entrapment, DNAs may enter cohesin rings through hinge opening. Mutation of three highly conserved lysine residues inside the Smc1 moiety of Smc1/3 hinges abolishes all loading without affecting cohesin's recruitment to CEN loading sites or its ability to hydrolyze ATP. We suggest that loading and translocation are mediated by conformational changes in cohesin's hinge driven by cycles of ATP hydrolysis.

| S-EPMC6371919 | biostudies-literature

Topological in vitro loading of the budding yeast cohesin ring onto DNA.

Project description:The ring-shaped chromosomal cohesin complex holds sister chromatids together by topological embrace, a prerequisite for accurate chromosome segregation. Cohesin plays additional roles in genome organization, transcriptional regulation and DNA repair. The cohesin ring includes an ABC family ATPase, but the molecular mechanism by which the ATPase contributes to cohesin function is not yet understood. Here we have purified budding yeast cohesin, as well as its Scc2-Scc4 cohesin loader complex, and biochemically reconstituted ATP-dependent topological cohesin loading onto DNA. Our results reproduce previous observations obtained using fission yeast cohesin, thereby establishing conserved aspects of cohesin behavior. Unexpectedly, we find that non-hydrolyzable ATP ground state mimetics ADP·BeF2, ADP·BeF3- and ADP·AlFx, but not a hydrolysis transition state analog ADP·VO43-, support cohesin loading. The energy from nucleotide binding is sufficient to drive the DNA entry reaction into the cohesin ring. ATP hydrolysis, thought to be essential for in vivo cohesin loading, must serve a subsequent reaction step. These results provide molecular insight into cohesin function and open new experimental opportunities that the budding yeast model affords.

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Project description:The Saccharomyces cerevisiae genome encodes five sirtuins (Sir2 and Hst1-4), which constitute a conserved family of NAD-dependent histone deacetylases. Cells lacking any individual sirtuin display mild growth and gene silencing defects. However, hst3Δ hst4Δ double mutants are exquisitely sensitive to genotoxins, and hst3Δ hst4Δ sir2Δmutants are inviable. Our published data also indicate that pharmacological inhibition of sirtuins prevents growth of several fungal pathogens, although the biological basis is unclear. Here, we present genome-wide fitness assays conducted with nicotinamide (NAM), a pan-sirtuin inhibitor. Our data indicate that NAM treatment causes yeast to solicit specific DNA damage response pathways for survival, and that NAM-induced growth defects are mainly attributable to inhibition of Hst3 and Hst4 and consequent elevation of histone H3 lysine 56 acetylation (H3K56ac). Our results further reveal that in the presence of constitutive H3K56ac, the Slx4 scaffolding protein and PP4 phosphatase complex play essential roles in preventing hyperactivation of the DNA damage-response kinase Rad53 in response to spontaneous DNA damage caused by reactive oxygen species. Overall, our data support the concept that chromosome-wide histone deacetylation by sirtuins is critical to mitigate growth defects caused by endogenous genotoxins.

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