Homologous recombination protects mammalian cells from replication-associated DNA double-strand breaks arising in response to methyl methanesulfonate

DNA-methylating agents of the S(N)2 type target DNA mostly at ring nitrogens, producing predominantly N-methylated purines These adducts are repaired by base excision repair(BER) Since defects in BER cause accumulation of DNA single-strand breaks (SSBs) and sensitize cells to the agents, it has been suggested that some of the lesions on their own or BER intermediates (e g apurinic sites) are cytotoxic, blocking DNA replication and inducing replication-mediated DNA double-strand breaks (DSBs) Here, we addressed the question of whether homologous recombination (HR) or non-homologous end-joining (NHEJ) or both are involved in the repair of DSBs formed following treatment of cells with methyl methanesulfonate (MMS) We show that HR defective cells (BRCA2, Rad51D and XRCC3 mutants) are dramatically more sensitive to MMS-Induced DNA damage as measured by colony formation, apoptosis and chromosomal aberrations, while NHEJ defective cells (Ku80 and DNA-PKCS mutants) are only mildly sensitive to the killing, apoptosis-inducing and clastogenic effects of MMS On the other hand, the HR mutants were almost completely refractory to the formation of sister chromatid exchanges (SCEs) following MMS treatment Since DSBs are expected to be formed specifically in the S-phase we assessed the formation and kinetics of repair of DSBs by gamma H2AX quantification in a cell cycle specific manner In the cytotoxic dose range of MMS a significant amount of gamma H2AX foci was Induced in S, but not G1- and G2-phase cells A major fraction of gamma H2AX foci colocalized with 53BP1 and phosphorylated ATM, indicating they are representative of DSBs DSB formation following MMS treatment was also demonstrated by the neutral comet assay Repair kinetics revealed that HR mutants exhibit a significant delay in DSB repair while NHEJ mutants completed S-phase specific DSB repair with a kinetic similar to the wildtype Moreover. DNA-PKcs inhibition in HR mutants did not affect the repair kinetics after MMS treatment Overall, the data indicate that agents producing N-alkylpurines in the DNA induce replication-dependent DSBs Further, they show that HR is the major pathway of protection of cells against DSB formation, killing and genotoxicity following S(N)2-alkylating agents (C) 2010 Elsevier B V All rights reserved