ABSTRACT
Histone H2AX plays a key role in DNA damage signalling in the surrounding regions of DNA double-strand breaks (DSBs). In response to DNA damage, H2AX becomes phosphorylated on serine residue 139 (known as γH2AX), resulting in the recruitment of the DNA repair effectors 53BP1 and BRCA1. Here, by studying resistance to poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1/2-deficient mammary tumours, we identify a function for γH2AX in orchestrating drug-induced replication fork degradation. Mechanistically, γH2AX-driven replication fork degradation is elicited by suppressing CtIP-mediated fork protection. As a result, H2AX loss restores replication fork stability and increases chemoresistance in BRCA1/2-deficient tumour cells without restoring homology-directed DNA repair, as highlighted by the lack of DNA damage-induced RAD51 foci. Furthermore, in the attempt to discover acquired genetic vulnerabilities, we find that ATM but not ATR inhibition overcomes PARP inhibitor (PARPi) resistance in H2AX-deficient tumours by interfering with CtIP-mediated fork protection. In summary, our results demonstrate a role for H2AX in replication fork biology in BRCA-deficient tumours and establish a function of H2AX separable from its classical role in DNA damage signalling and DSB repair.
Subject(s)
BRCA1 Protein , BRCA2 Protein , DNA Replication , Drug Resistance, Neoplasm , Histones , Poly(ADP-ribose) Polymerase Inhibitors , Animals , Female , Humans , Mice , Ataxia Telangiectasia Mutated Proteins/metabolism , Ataxia Telangiectasia Mutated Proteins/genetics , BRCA1 Protein/metabolism , BRCA1 Protein/deficiency , BRCA1 Protein/genetics , BRCA2 Protein/metabolism , BRCA2 Protein/genetics , BRCA2 Protein/deficiency , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Breast Neoplasms/drug therapy , Carrier Proteins/metabolism , Carrier Proteins/genetics , Cell Line, Tumor , DNA Breaks, Double-Stranded , DNA Damage , DNA Repair , DNA Replication/drug effects , Drug Resistance, Neoplasm/genetics , Histones/metabolism , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Rad51 Recombinase/metabolism , Rad51 Recombinase/genetics , Tumor Suppressor p53-Binding Protein 1/metabolism , Tumor Suppressor p53-Binding Protein 1/genetics , Mice, NudeABSTRACT
DNA double-strand break (DSB) repair is initiated by DNA end resection. CtIP acts in short-range resection to stimulate MRE11-RAD50-NBS1 (MRN) to endonucleolytically cleave 5'-terminated DNA to bypass protein blocks. CtIP also promotes the DNA2 helicase-nuclease to accelerate long-range resection downstream from MRN. Here, using AlphaFold2, we identified CtIP-F728E-Y736E as a separation-of-function mutant that is still proficient in conjunction with MRN but is not able to stimulate ssDNA degradation by DNA2. Accordingly, CtIP-F728E-Y736E impairs physical interaction with DNA2. Cellular assays revealed that CtIP-F728E-Y736E cells exhibit reduced DSB-dependent chromatin-bound RPA, impaired long-range resection, and increased sensitivity to DSB-inducing drugs. Previously, CtIP was shown to be targeted by PLK1 to inhibit long-range resection, yet the underlying mechanism was unclear. We show that the DNA2-interacting region in CtIP includes the PLK1 target site at S723. The integrity of S723 in CtIP is necessary for the stimulation of DNA2, and phosphorylation of CtIP by PLK1 in vitro is consequently inhibitory, explaining why PLK1 restricts long-range resection. Our data support a model in which CDK-dependent phosphorylation of CtIP activates resection by MRN in S phase, and PLK1-mediated phosphorylation of CtIP disrupts CtIP stimulation of DNA2 to attenuate long-range resection later at G2/M.
