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Local activation of mammalian separase in interphase promotes double strand break repair and prevents oncogenic transformation

ABSTRACT: Separase halves eukaryotic chromosomes in M-phase by cleaving cohesin complexes holding sister chromatids together. Whether this essential protease functions also in interphase and/or impacts carcinogenesis remains largely unknown. Here, we show that mammalian separase is recruited to DNA double strand breaks (DSBs) where it is activated to locally cleave cohesin and facilitate homology directed repair (HDR). Inactivating phosphorylation of its NES, arginine methylation of its RG-repeats, and sumoylation redirect separase from the cytosol to DSBs. In vitro assays suggest that DNA damage response-relevant ATM, PRMT1 and Mms21 represent the corresponding kinase, methyltransferase and SUMO-ligase, respectively. SEPARASE heterozygosity not only debilitates HDR but also predisposes primary embryonic fibroblasts to neoplasia and mice to chemically-induced skin cancer. Thus, tethering of separase to DSBs and confined cohesin cleavage promote DSB repair in G2 cells. Importantly, this conserved interphase function of separase protects mammalian cells from oncogenic transformation.-

SUBMITTER: Dr. Olaf Stemmann

PROVIDER: S-SCDT-EMBOJ-2018-99184P | biostudies-other |

REPOSITORIES: biostudies-other

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Local activation of mammalian separase in interphase promotes double-strand break repair and prevents oncogenic transformation.

Project description:Separase halves eukaryotic chromosomes in M-phase by cleaving cohesin complexes holding sister chromatids together. Whether this essential protease functions also in interphase and/or impacts carcinogenesis remains largely unknown. Here, we show that mammalian separase is recruited to DNA double-strand breaks (DSBs) where it is activated to locally cleave cohesin and facilitate homology-directed repair (HDR). Inactivating phosphorylation of its NES, arginine methylation of its RG-repeats, and sumoylation redirect separase from the cytosol to DSBs. In vitro assays suggest that DNA damage response-relevant ATM, PRMT1, and Mms21 represent the corresponding kinase, methyltransferase, and SUMO ligase, respectively. SEPARASE heterozygosity not only debilitates HDR but also predisposes primary embryonic fibroblasts to neoplasia and mice to chemically induced skin cancer. Thus, tethering of separase to DSBs and confined cohesin cleavage promote DSB repair in G2 cells. Importantly, this conserved interphase function of separase protects mammalian cells from oncogenic transformation.

| S-EPMC6236338 | biostudies-literature

Local activation of mammalian separase in interphase promotes double strand break repair and prevents oncogenic transformation

Project description:Separase halves eukaryotic chromosomes in M-phase by cleaving cohesin complexes holding sister chromatids together. Whether this essential protease functions also in interphase and/or impacts carcinogenesis remains largely unknown. Here, we show that mammalian separase is recruited to DNA double strand breaks (DSBs) where it is activated to locally cleave cohesin and facilitate homology directed repair (HDR). Inactivating phosphorylation of its NES, arginine methylation of its RG-repeats, and sumoylation redirect separase from the cytosol to DSBs. In vitro assays suggest that DNA damage response-relevant ATM, PRMT1 and Mms21 represent the corresponding kinase, methyltransferase and SUMO-ligase, respectively. SEPARASE heterozygosity not only debilitates HDR but also predisposes primary embryonic fibroblasts to neoplasia and mice to chemically-induced skin cancer. Thus, tethering of separase to DSBs and confined cohesin cleavage promote DSB repair in G2 cells. Importantly, this conserved interphase function of separase protects mammalian cells from oncogenic transformation.-

| S-SCDT-EMBOJ-2018-99184 | biostudies-other

Branch migration prevents DNA loss during double-strand break repair.

Project description:The repair of DNA double-strand breaks must be accurate to avoid genomic rearrangements that can lead to cell death and disease. This can be accomplished by promoting homologous recombination between correctly aligned sister chromosomes. Here, using a unique system for generating a site-specific DNA double-strand break in one copy of two replicating Escherichia coli sister chromosomes, we analyse the intermediates of sister-sister double-strand break repair. Using two-dimensional agarose gel electrophoresis, we show that when double-strand breaks are formed in the absence of RuvAB, 4-way DNA (Holliday) junctions are accumulated in a RecG-dependent manner, arguing against the long-standing view that the redundancy of RuvAB and RecG is in the resolution of Holliday junctions. Using pulsed-field gel electrophoresis, we explain the redundancy by showing that branch migration catalysed by RuvAB and RecG is required for stabilising the intermediates of repair as, when branch migration cannot take place, repair is aborted and DNA is lost at the break locus. We demonstrate that in the repair of correctly aligned sister chromosomes, an unstable early intermediate is stabilised by branch migration. This reliance on branch migration may have evolved to help promote recombination between correctly aligned sister chromosomes to prevent genomic rearrangements.

| S-EPMC4125073 | biostudies-literature

Subtelomeric regions in mammalian cells are deficient in DNA double-strand break repair.

Project description:We have previously demonstrated that double-strand breaks (DSBs) in regions near telomeres are much more likely to result in large deletions, gross chromosome rearrangements, and chromosome instability than DSBs at interstitial sites within chromosomes. In the present study, we investigated whether this response of subtelomeric regions to DSBs is a result of a deficiency in DSB repair by comparing the frequency of homologous recombination repair (HRR) and nonhomologous end joining (NHEJ) at interstitial and telomeric sites following the introduction of DSBs by I-SceI endonuclease. We also monitored the frequency of small deletions, which have been shown to be the most common mutation at I-SceI-induced DSBs at interstitial sites. We observed no difference in the frequency of small deletions or HRR at interstitial and subtelomeric DSBs. However, the frequency of NHEJ was significantly lower at DSBs near telomeres compared to interstitial sites. The frequency of NHEJ was also lower at DSBs occurring at interstitial sites containing telomeric repeat sequences. We propose that regions near telomeres are deficient in classical NHEJ as a result of the presence of cis-acting telomere-binding proteins that cause DSBs to be processed as though they were telomeres, resulting in excessive resection, telomere loss, and eventual chromosome rearrangements by alternative NHEJ.

| S-EPMC3084351 | biostudies-literature

Mammalian SWI/SNF complexes facilitate DNA double-strand break repair by promoting gamma-H2AX induction.

Project description:Although mammalian SWI/SNF chromatin remodeling complexes have been well established to play important role in transcription, their role in DNA repair has remained largely unexplored. Here we show that inactivation of the SWI/SNF complexes and downregulation of the catalytic core subunits of the complexes both result in inefficient DNA double-strand break (DSB) repair and increased DNA damage sensitivity as well as a large defect in H2AX phosphorylation (gamma-H2AX) and nuclear focus formation after DNA damage. The expression of most DSB repair genes remains unaffected and DNA damage checkpoints are grossly intact in the cells inactivated for the SWI/SNF complexes. Although the SWI/SNF complexes do not affect the expression of ATM, DNA-PK and ATR, or their activation and/or recruitment to DSBs, they rapidly bind to DSB-surrounding chromatin via interaction with gamma-H2AX in the manner that is dependent on the amount of DNA damage. Given the crucial role for gamma-H2AX in efficient DSB repair, these results suggest that the SWI/SNF complexes facilitate DSB repair, at least in part, by promoting H2AX phosphorylation by directly acting on chromatin.

| S-EPMC1560357 | biostudies-literature

Distinct roles of chromatin-associated proteins MDC1 and 53BP1 in mammalian double-strand break repair.

Project description:Phosphorylated histone H2AX ("gamma-H2AX") recruits MDC1, 53BP1, and BRCA1 to chromatin near a double-strand break (DSB) and facilitates efficient repair of the break. It is unclear to what extent gamma-H2AX-associated proteins act in concert and to what extent their functions within gamma-H2AX chromatin are distinct. We addressed this question by comparing the mechanisms of action of MDC1 and 53BP1 in DSB repair (DSBR). We find that MDC1 functions primarily in homologous recombination/sister chromatid recombination, in a manner strictly dependent upon its ability to interact with gamma-H2AX but, unexpectedly, not requiring recruitment of 53BP1 or BRCA1 to gamma-H2AX chromatin. In contrast, 53BP1 functions in XRCC4-dependent nonhomologous end-joining, likely mediated by its interaction with dimethylated lysine 20 of histone H4 but, surprisingly, independent of H2AX. These results suggest a specialized adaptation of the "histone code" in which distinct histone tail-protein interactions promote engagement of distinct DSBR pathways.

| S-EPMC2275782 | biostudies-literature

Contribution of telomerase RNA retrotranscription to DNA double-strand break repair during mammalian genome evolution.

Project description:BackgroundIn vertebrates, tandem arrays of TTAGGG hexamers are present at both telomeres and intrachromosomal sites (interstitial telomeric sequences (ITSs)). We previously showed that, in primates, ITSs were inserted during the repair of DNA double-strand breaks and proposed that they could arise from either the capture of telomeric fragments or the action of telomerase.ResultsAn extensive comparative analysis of two primate (Homo sapiens and Pan troglodytes) and two rodent (Mus musculus and Rattus norvegicus) genomes allowed us to describe organization and insertion mechanisms of all the informative ITSs present in the four species. Two novel observations support the hypothesis of telomerase involvement in ITS insertion: in a highly significant fraction of informative loci, the ITSs were introduced at break sites where a few nucleotides homologous to the telomeric hexamer were exposed; in the rodent genomes, complex ITS loci are present in which a retrotranscribed fragment of the telomerase RNA, far away from the canonical template, was inserted together with the telomeric repeats. Moreover, mutational analysis of the TTAGGG arrays in the different species suggests that they were inserted as exact telomeric hexamers, further supporting the participation of telomerase in ITS formation.ConclusionThese results strongly suggest that telomerase was utilized, in some instances, for the repair of DNA double-strand breaks occurring in the genomes of rodents and primates during evolution. The presence, in the rodent genomes, of sequences retrotranscribed from the telomerase RNA strengthens the hypothesis of the origin of telomerase from an ancient retrotransposon.

| S-EPMC2246262 | biostudies-literature

A model of oncogenic rearrangements: differences between chromosomal translocation mechanisms and simple double-strand break repair.

Project description:Recurrent reciprocal translocations are present in many hematologic and mesenchymal malignancies. Because significant sequence homology is absent from translocation breakpoint junctions, non-homologous end-joining (NHEJ) pathways of DNA repair are presumed to catalyze their formation. We developed translocation reporters for use in mammalian cells from which NHEJ events can be selected after precise chromosomal breakage. Translocations were efficiently recovered with these reporters using mouse cells, and their breakpoint junctions recapitulated findings from oncogenic translocations. Small deletions and microhomology were present in most junctions; insertions and more complex events also were observed. Thus, our reporters model features of oncogenic rearrangements in human cancer cells. A homologous sequence at a distance from the break site affected the translocation junction without substantially altering translocation frequency. Interestingly, in a direct comparison, the spectrum of translocation breakpoint junctions differed from junctions derived from repair at a single chromosomal break, providing mechanistic insight into translocation formation.

| S-EPMC1895622 | biostudies-literature

FKBP25 participates in DNA double-strand break repair.

Project description:FK506-binding proteins (FKBPs) alter the conformation of proteins via cis-trans isomerization of prolyl-peptide bonds. While this activity can be demonstrated in vitro, the intractability of detecting prolyl isomerization events in cells has limited our understanding of the biological processes regulated by FKBPs. Here we report that FKBP25 is an active participant in the repair of DNA double-strand breaks (DSBs). FKBP25 influences DSB repair pathway choice by promoting homologous recombination (HR) and suppressing single-strand annealing (SSA). Consistent with this observation, cells depleted of FKBP25 form fewer Rad51 repair foci in response to etoposide and ionizing radiation, and they are reliant on the SSA repair factor Rad52 for viability. We find that FKBP25's catalytic activity is required for promoting DNA repair, which is the first description of a biological function for this enzyme activity. Consistent with the importance of the FKBP catalytic site in HR, rapamycin treatment also impairs homologous recombination, and this effect is at least in part independent of mTor. Taken together these results identify FKBP25 as a component of the DNA DSB repair pathway.

| S-EPMC7457334 | biostudies-literature

Pathological mutations in PNKP trigger defects in DNA single-strand break repair but not DNA double-strand break repair.

Project description:Hereditary mutations in polynucleotide kinase-phosphatase (PNKP) result in a spectrum of neurological pathologies ranging from neurodevelopmental dysfunction in microcephaly with early onset seizures (MCSZ) to neurodegeneration in ataxia oculomotor apraxia-4 (AOA4) and Charcot-Marie-Tooth disease (CMT2B2). Consistent with this, PNKP is implicated in the repair of both DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs); lesions that can trigger neurodegeneration and neurodevelopmental dysfunction, respectively. Surprisingly, however, we did not detect a significant defect in DSB repair (DSBR) in primary fibroblasts from PNKP patients spanning the spectrum of PNKP-mutated pathologies. In contrast, the rate of SSB repair (SSBR) is markedly reduced. Moreover, we show that the restoration of SSBR in patient fibroblasts collectively requires both the DNA kinase and DNA phosphatase activities of PNKP, and the fork-head associated (FHA) domain that interacts with the SSBR protein, XRCC1. Notably, however, the two enzymatic activities of PNKP appear to affect different aspects of disease pathology, with reduced DNA phosphatase activity correlating with neurodevelopmental dysfunction and reduced DNA kinase activity correlating with neurodegeneration. In summary, these data implicate reduced rates of SSBR, not DSBR, as the source of both neurodevelopmental and neurodegenerative pathology in PNKP-mutated disease, and the extent and nature of this reduction as the primary determinant of disease severity.

| S-EPMC7337934 | biostudies-literature

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