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BRCA1 deficiency specific base substitution mutagenesis is dependent on translesion synthesis and regulated by 53BP1.

ABSTRACT: Defects in BRCA1, BRCA2 and other genes of the homology-dependent DNA repair (HR) pathway cause an elevated rate of mutagenesis, eliciting specific mutation patterns including COSMIC signature SBS3. Using genome sequencing of knock-out cell lines we show that Y family translesion synthesis (TLS) polymerases contribute to the spontaneous generation of base substitution and short insertion/deletion mutations in BRCA1 deficient cells, and that TLS on DNA adducts is increased in BRCA1 and BRCA2 mutants. The inactivation of 53BP1 in BRCA1 mutant cells markedly reduces TLS-specific mutagenesis, and rescues the deficiency of template switch-mediated gene conversions in the immunoglobulin V locus of BRCA1 mutant chicken DT40 cells. 53BP1 also promotes TLS in human cellular extracts in vitro. Our results show that HR deficiency-specific mutagenesis is largely caused by TLS, and suggest a function for 53BP1 in regulating the choice between TLS and error-free template switching in replicative DNA damage bypass.

SUBMITTER: Chen D

PROVIDER: S-EPMC8752635 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Translesion DNA synthesis and mutagenesis in eukaryotes.

Project description:The structural features that enable replicative DNA polymerases to synthesize DNA rapidly and accurately also limit their ability to copy damaged DNA. Direct replication of DNA damage is termed translesion synthesis (TLS), a mechanism conserved from bacteria to mammals and executed by an array of specialized DNA polymerases. This chapter examines how these translesion polymerases replicate damaged DNA and how they are regulated to balance their ability to replicate DNA lesions with the risk of undesirable mutagenesis. It also discusses how TLS is co-opted to increase the diversity of the immunoglobulin gene hypermutation and the contribution it makes to the mutations that sculpt the genome of cancer cells.

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Loss of BRCA1 or BRCA2 markedly increases the rate of base substitution mutagenesis and has distinct effects on genomic deletions.

Project description:Loss-of-function mutations in the BRCA1 and BRCA2 genes increase the risk of cancer. Owing to their function in homologous recombination repair, much research has focused on the unstable genomic phenotype of BRCA1/2 mutant cells manifest mainly as large-scale rearrangements. We used whole-genome sequencing of multiple isogenic chicken DT40 cell clones to precisely determine the consequences of BRCA1/2 loss on all types of genomic mutagenesis. Spontaneous base substitution mutation rates increased sevenfold upon the disruption of either BRCA1 or BRCA2, and the arising mutation spectra showed strong and specific correlation with a mutation signature associated with BRCA1/2 mutant tumours. To model endogenous alkylating damage, we determined the mutation spectrum caused by methyl methanesulfonate (MMS), and showed that MMS also induces more base substitution mutations in BRCA1/2-deficient cells. Spontaneously arising and MMS-induced insertion/deletion mutations and large rearrangements were also more common in BRCA1/2 mutant cells compared with the wild-type control. A difference in the short deletion phenotypes of BRCA1 and BRCA2 suggested distinct roles for the two proteins in the processing of DNA lesions, as BRCA2 mutants contained more short deletions, with a wider size distribution, which frequently showed microhomology near the breakpoints resembling repair by non-homologous end joining. An increased and prolonged gamma-H2AX signal in MMS-treated BRCA1/2 cells suggested an aberrant processing of stalled replication forks as the cause of increased mutagenesis. The high rate of base substitution mutagenesis demonstrated by our experiments is likely to significantly contribute to the oncogenic effect of the inactivation of BRCA1 or BRCA2.

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Project description:The efficacy of poly(ADP)-ribose polymerase 1 inhibition (PARPi) in BRCA1-deficient cells depends on 53BP1 and shieldin, which have been proposed to limit single-stranded DNA at double-strand breaks (DSBs) by blocking resection and/or through CST-Polα-primase-mediated fill-in. We show that primase (like 53BP1-shieldin and CST-Polα) promotes radial chromosome formation in PARPi-treated BRCA1-deficient cells and demonstrate shieldin-CST-Polα-primase-dependent incorporation of BrdU at DSBs. In the absence of 53BP1 or shieldin, radial formation in BRCA1-deficient cells was restored by the tethering of CST near DSBs, arguing that in this context, shieldin acts primarily by recruiting CST. Furthermore, a SHLD1 mutant defective in CST binding (SHLD1Δ) was non-functional in BRCA1-deficient cells and its function was restored after reconnecting SHLD1Δ to CST. Interestingly, at dysfunctional telomeres and at DNA breaks in class switch recombination where CST has been implicated, SHLD1Δ was fully functional, perhaps because these DNA ends carry CST recognition sites that afford SHLD1-independent binding of CST. These data establish that in BRCA1-deficient cells, CST-Polα-primase is the major effector of shieldin-dependent DSB processing.

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Translesion DNA synthesis-driven mutagenesis in very early embryogenesis of fast cleaving embryos

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2021-12-28 | GSE161335 | GEO

Cell cycle-dependent inhibition of 53BP1 signaling by BRCA1.

Project description:DNA damage response mediator protein 53BP1 is a key regulator of non-homologous end-joining (NHEJ) repair. 53BP1 protects DNA broken ends from resection by recruiting two downstream factors, RIF1 (RAP1-interacting factor 1) and PTIP (Pax transactivation domain-interacting protein), to double-stranded breaks (DSBs) via ATM (ataxia telangiectasia mutated)-mediated 53BP1 phosphorylation, and competes with BRCA1-mediated homologous recombination (HR) repair in G1 phase. In contrast, BRCA1 antagonizes 53BP1-direct NHEJ repair in S/G2 phases. We and others have found that BRCA1 prevents the translocation of RIF1 to DSBs in S/G2 phases; however, the underlying mechanism remains unclear. Here we show that efficient ATM-dependent 53BP1 phosphorylation is restricted to the G1 phase of the cell cycle, as a consequence RIF1 and PTIP accumulation at DSB sites only occur in G1 phase. Mechanistically, both BRCT and RING domains of BRCA1 are required for the inhibition of 53BP1 phosphorylation in S and G2 phases. Thus, our findings reveal how BRCA1 antagonizes 53BP1 signaling to ensure that HR repair is the dominant repair pathway in S/G2 phases.

| S-EPMC4860855 | biostudies-other

RecFOR proteins are essential for Pol V-mediated translesion synthesis and mutagenesis.

Project description:When the replication fork moves through the template DNA containing lesions, daughter-strand gaps are formed opposite lesion sites. These gaps are subsequently filled-in either by translesion synthesis (TLS) or by homologous recombination. RecA filaments formed within these gaps are key intermediates for both of the gap-filling pathways. For instance, Pol V, the major lesion bypass polymerase in Escherichia coli, requires a functional interaction with the tip of the RecA filament. Here, we show that all three recombination mediator proteins RecFOR are needed to build a functionally competent RecA filament that supports efficient Pol V-mediated TLS in the presence of ssDNA-binding protein (SSB). A positive contribution of RecF protein to Pol V lesion bypass is demonstrated. When Pol III and Pol V are both present, Pol III imparts a negative effect on Pol V-mediated lesion bypass that is counteracted by the combined action of RecFOR and SSB. Mutations in recF, recO or recR gene abolish induced mutagenesis in E. coli.

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Eukaryotic stress-induced mutagenesis is limited by a local control of translesion synthesis.

Project description:The DNA damage response (DDR) preserves the genetic integrity of the cell by sensing and repairing damages after a genotoxic stress. Translesion Synthesis (TLS), an error-prone DNA damage tolerance pathway, is controlled by PCNA ubiquitination. In this work, we raise the question whether TLS is controlled locally or globally. Using a recently developed method that allows to follow the bypass of a single lesion inserted into the yeast genome, we show that (i) TLS is controlled locally at each individual lesion by PCNA ubiquitination, (ii) a single lesion is enough to induce PCNA ubiquitination and (iii) PCNA ubiquitination is imperative for TLS to occur. More importantly, we show that the activation of the DDR that follows a genotoxic stress does not increase TLS at individual lesions. We conclude that unlike the SOS response in bacteria, the eukaryotic DDR does not promote TLS and mutagenesis.

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Translesion DNA synthesis-driven mutagenesis in very early embryogenesis of fast cleaving embryos.

Project description:In early embryogenesis of fast cleaving embryos, DNA synthesis is short and surveillance mechanisms preserving genome integrity are inefficient, implying the possible generation of mutations. We have analyzed mutagenesis in Xenopus laevis and Drosophila melanogaster early embryos. We report the occurrence of a high mutation rate in Xenopus and show that it is dependent upon the translesion DNA synthesis (TLS) master regulator Rad18. Unexpectedly, we observed a homology-directed repair contribution of Rad18 in reducing the mutation load. Genetic invalidation of TLS in the pre-blastoderm Drosophila embryo resulted in reduction of both the hatching rate and single-nucleotide variations on pericentromeric heterochromatin in adult flies. Altogether, these findings indicate that during very early Xenopus and Drosophila embryos TLS strongly contributes to the high mutation rate. This may constitute a previously unforeseen source of genetic diversity contributing to the polymorphisms of each individual with implications for genome evolution and species adaptation.

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A selective requirement for 53BP1 in the biological response to genomic instability induced by Brca1 deficiency.

Project description:The molecular pathways leading from genomic instability to cellular senescence and/or cell death remain incompletely characterized. Using mouse embryonic fibroblasts with constitutively increased DNA damage due to the absence of the full-length form of the tumor suppressor Brca1 (Brca1(Delta 11/Delta 11)), we show that deletion of p53 binding protein 1 (53BP1) selectivity abrogates senescence and cell death stimulated by reduced Brca1 activity. Furthermore, the embryonic lethality induced by Brca1 mutation can be alleviated by 53BP1 deletion. Adult Brca1(Delta 11/Delta 11)53BP1(-/-) manifest constitutively high levels of genomic instability, yet age relatively normally, with a surprisingly low incidence of overall tumor formation. Together, these in vitro and in vivo data suggest that 53BP1 is specifically required for the development of premature senescence and apoptosis induced by Brca1 deficiency. These observations may have important implications for Brca1-mediated tumor formation as well as for the molecular pathway leading from genomic instability to organismal aging.

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53BP1 loss rescues BRCA1 deficiency and is associated with triple-negative and BRCA-mutated breast cancers.

Project description:Germ-line mutations in breast cancer 1, early onset (BRCA1) result in predisposition to breast and ovarian cancer. BRCA1-mutated tumors show genomic instability, mainly as a consequence of impaired recombinatorial DNA repair. Here we identify p53-binding protein 1 (53BP1) as an essential factor for sustaining the growth arrest induced by Brca1 deletion. Depletion of 53BP1 abrogates the ATM-dependent checkpoint response and G2 cell-cycle arrest triggered by the accumulation of DNA breaks in Brca1-deleted cells. This effect of 53BP1 is specific to BRCA1 function, as 53BP1 depletion did not alleviate proliferation arrest or checkpoint responses in Brca2-deleted cells. Notably, loss of 53BP1 partially restores the homologous-recombination defect of Brca1-deleted cells and reverts their hypersensitivity to DNA-damaging agents. We find reduced 53BP1 expression in subsets of sporadic triple-negative and BRCA-associated breast cancers, indicating the potential clinical implications of our findings.

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