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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.

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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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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.

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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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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.

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

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Replicated chromatin curtails 53BP1 recruitment in BRCA1-proficient and BRCA1-deficient cells.

Project description:DNA double-strand breaks can be repaired by non-homologous end-joining or homologous recombination. Which pathway is used depends on the balance between the tumor suppressors 53BP1 and BRCA1 and on the availability of an undamaged template DNA for homology-directed repair. How cells switch from a 53BP1-dominated to a BRCA1-governed homologous recombination response as they progress through the cell cycle is incompletely understood. Here we reveal, using high-throughput microscopy and applying single cell normalization to control for increased genome size as cells replicate their DNA, that 53BP1 recruitment to damaged replicated chromatin is inefficient in both BRCA1-proficient and BRCA1-deficient cells. Our results substantiate a dual switch model from a 53BP1-dominated response in unreplicated chromatin to a BRCA1-BARD1-dominated response in replicated chromatin, in which replication-coupled dilution of 53BP1's binding mark H4K20me2 functionally cooperates with BRCA1-BARD1-mediated suppression of 53BP1 binding. More generally, we suggest that appropriate normalization of single cell data, for example, to DNA content, provides additional layers of information, which can be critical for quantifying and interpreting cellular phenotypes.

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