RESUMO
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.
Assuntos
Proteína BRCA1 , Proteína BRCA2 , Replicação do DNA , Resistencia a Medicamentos Antineoplásicos , Histonas , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Feminino , Humanos , Camundongos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteína BRCA1/metabolismo , Proteína BRCA1/deficiência , Proteína BRCA1/genética , Proteína BRCA2/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/deficiência , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Reparo do DNA , Replicação do DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Histonas/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Rad51 Recombinase/metabolismo , Rad51 Recombinase/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Camundongos NusRESUMO
Template-directed CRISPR/Cas9 editing is a powerful tool for introducing subtle mutations in genomes. However, the success rate of incorporation of the desired mutations at the target site is difficult to predict and therefore must be empirically determined. Here, we adapted the widely used TIDE method for quantification of templated editing events, including point mutations. The resulting TIDER method is a rapid, cheap and accessible tool for testing and optimization of template-directed genome editing strategies. A free web tool for TIDER data analysis is available at http://tide.nki.nl.
