Mismatch repair-dependent processing of methylation damage gives rise to persistent single-stranded gaps in newly replicated DNA

Abstract

O6-Methylguanine (MeG) is a highly cytotoxic DNA modification generated by SN1-type methylating agents. Despite numerous studies implicating DNA replication, mismatch repair (MMR), and homologous recombination (HR) in MeG toxicity, its mode of action has remained elusive. We studied the molecular transactions in the DNA of yeast and mammalian cells treated with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Although replication fork progression was unaffected in the first cell cycle after treatment, electron microscopic analysis revealed an accumulation of MeG- and MMR-dependent single-stranded DNA (ssDNA) gaps in newly replicated DNA. Progression into the second cell cycle required HR, while the following G2 arrest required the continued presence of MeG. Yeast cells overcame this block, while mammalian cells generally failed to recover, and those that did contained multiple sister chromatid exchanges. Notably, the arrest could be abolished by removal of MeG after the first S phase. These new data provide compelling support for the hypothesis that MMR attempts to correct MeG/C or MeG/T mispairs arising during replication. Due to the persistence of MeG in the exposed template strand, repair synthesis cannot take place, which leaves single-stranded gaps behind the replication fork. During the subsequent S phase, these gaps cause replication fork collapse and elicit recombination and cell cycle arrest.