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BRCA1/2 mutant tumor cells display an elevated mutation burden, the etiology of which remains unclear. Here, we report that these cells accumulate ssDNA gaps and spontaneous mutations during unperturbed DNA replication due to repriming by the DNA primase-polymerase PRIMPOL. Gap accumulation requires the DNA glycosylase SMUG1 and is exacerbated by depletion of the translesion synthesis (TLS) factor RAD18 or inhibition of the error-prone TLS polymerase complex REV1-Polζ by the small molecule JH-RE-06. JH-RE-06 treatment of BRCA1/2-deficient cells results in reduced mutation rates and PRIMPOL- and SMUG1-dependent loss of viability. Through cellular and animal studies, we demonstrate that JH-RE-06 is preferentially toxic toward HR-deficient cancer cells. Furthermore, JH-RE-06 remains effective toward PARP inhibitor (PARPi)-resistant BRCA1 mutant cells and displays additive toxicity with crosslinking agents or PARPi. Collectively, these studies identify a protective and mutagenic role for REV1-Polζ in BRCA1/2 mutant cells and provide the rationale for using REV1-Polζ inhibitors to treat BRCA1/2 mutant tumors. [Display omitted] • ssDNA gaps arise in BRCA1 mutant cancer cells due to PRIMPOL-mediated repriming • BRCA1/2 deficiency leads to mutagenic ssDNA gap repair by REV1-Polζ-dependent TLS • Targeted REV1-Polζ inhibition shows enhanced toxicity in HR-deficient cancer cells • ssDNA gaps formed by SMUG1 and PRIMPOL mediate the toxicity of REV1-Polζ inhibition Taglialatela et al. report that homologous recombination (HR)-deficient cancer cells, such as BRCA1/2 mutant cells, display increased reliance on error-prone translesion synthesis (TLS) for the repair of ssDNA gaps arising spontaneously during DNA replication. TLS inhibition shows exquisite toxicity in BRCA1/2-deficient cancer cells, providing the basis for alternative therapies against BRCA1/2 mutant tumors. [ABSTRACT FROM AUTHOR]

Stabilization of stalled replication forks is a prominent mechanism of PARP (Poly(ADP-ribose) Polymerase) inhibitor (PARPi) resistance in BRCA -deficient tumors. Epigenetic mechanisms of replication fork stability are emerging but remain poorly understood. Here, we report the histone acetyltransferase PCAF (p300/CBP-associated) as a fork-associated protein that promotes fork degradation in BRCA-deficient cells by acetylating H4K8 at stalled replication forks, which recruits MRE11 and EXO1. A H4K8ac binding domain within MRE11/EXO1 is required for their recruitment to stalled forks. Low PCAF levels, which we identify in a subset of BRCA2 -deficient tumors, stabilize stalled forks, resulting in PARPi resistance in BRCA-deficient cells. Furthermore, PCAF activity is tightly regulated by ATR (ataxia telangiectasia and Rad3-related), which phosphorylates PCAF on serine 264 (S264) to limit its association and activity at stalled forks. Our results reveal PCAF and histone acetylation as critical regulators of fork stability and PARPi responses in BRCA-deficient cells, which provides key insights into targeting BRCA -deficient tumors and identifying epigenetic modulators of chemotherapeutic responses. • Loss of PCAF results in fork protection and PARPi resistance in BRCA-deficient cells • PCAF acetylates histone H4 at lysine 8 at stalled replication forks • MRE11 and EXO1 bind to H4K8ac at stalled replication forks in BRCA-deficient cells • PCAF is phosphorylated by ATR at S264, which limits PCAF functions at stalled forks Kim et al. demonstrate replication fork association of the histone acetyltransferase PCAF, which promotes replication fork degradation in BRCA-deficient cells. The nucleases MRE11 and EXO1 bind PCAF-mediated H4K8ac at stalled forks. PCAF loss confers PARP inhibitor resistance in BRCA-deficient cells, identifying PCAF and H4K8ac in chemotherapeutic responses and fork stability. [ABSTRACT FROM AUTHOR]

Summary To ensure the completion of DNA replication and maintenance of genome integrity, DNA repair factors protect stalled replication forks upon replication stress. Previous studies have identified a critical role for the tumor suppressors BRCA1 and BRCA2 in preventing the degradation of nascent DNA by the MRE11 nuclease after replication stress. Here we show that depletion of SMARCAL1, a SNF2-family DNA translocase that remodels stalled forks, restores replication fork stability and reduces the formation of replication stress-induced DNA breaks and chromosomal aberrations in BRCA1/2-deficient cells. In addition to SMARCAL1, other SNF2-family fork remodelers, including ZRANB3 and HLTF, cause nascent DNA degradation and genomic instability in BRCA1/2-deficient cells upon replication stress. Our observations indicate that nascent DNA degradation in BRCA1/2-deficient cells occurs as a consequence of MRE11-dependent nucleolytic processing of reversed forks generated by fork remodelers. These studies provide mechanistic insights into the processes that cause genome instability in BRCA1/2-deficient cells. [ABSTRACT FROM AUTHOR]