Processing of O6-methylguanine into DNA double-strand breaks requires two rounds of replication whereas apoptosis is also induced in subsequent cell cycles

Abstract

The DNA adduct O6-methylguanine (O6MeG) induced by environmental genotoxins and anticancer drugs is a highly mutagenic, genotoxic and apoptotic lesion. Apoptosis induced by O6MeG requires mismatch repair (MMR) and proliferation. Models of O6MeG-triggered cell death postulate that O6MeG/T mispairs activate MMR giving rise to either direct genotoxic signaling or secondary lesions that trigger apoptotic signaling in the 2nd replication cycle. To test these hypotheses, we used a highly synchronized cell system competent and deficient for the repair of O6MeG adducts, which were induced by the SN1 methylating agent N-methyl-N’-nitro-N-nitrosoguanidine (MNNG). We show that DNA double-strand breaks (DSBs) are formed in response to O6MeG at high level in the 2nd S/G2-phase of the cell cycle. This is accompanied by ATR and Chk1 phosphorylation, G2/M arrest and late caspase activation. Although cells undergo apoptosis out of the 2nd G2/M-phase, the majority of them recovers and undergoes apoptosis after passing through additional replication cycles. The late apoptotic effects were completely abolished by O6-methylguanine-DNA methyltransferase, indicating that non-repaired O6MeG is carried over into subsequent generations, eliciting there a late apoptotic response. We also demonstrate that with a low, non-toxic dose of MNNG the passage of cells through the 1st and 2nd S-phase is not delayed, although the dose is able to induce excessive sister chromatid exchanges. This suggests that a significant amount of O6MeG can be tolerated by recombination, which is a fast process preventing from S-phase blockage, DSB formation and cell death.