Repair of O6-methylguanine is not affected by thymine base pairing and the presence of MMR proteins

Abstract

Methylation at the O6-position of guanine (O6-MeG) by alkylating agents is efficiently removed by O6-methylguanine–DNA methyltransferase (MGMT), preventing from cytotoxic, mutagenic, clastogenic and carcinogenic effects of O6-MeG-inducing agents. If O6-MeG is not removed from DNA prior to replication, thymine will be incorporated instead of cytosine opposite the O6-MeG lesion. This mismatch is recognized and processed by mismatch repair (MMR) proteins which are known to be involved in triggering the cytotoxic and genotoxic response of cells upon methylation. In this work we addressed three open questions. (1) Is MGMT able to repair O6-MeG mispaired with thymine (O6-MeG/T)? (2) Do MMR proteins interfere with the repair of O6-MeG/T by MGMT? (3) Does MGMT show a protective effect if it is expressed after replication of DNA containing O6-MeG? Using an in vitro assay we show that oligonucleotides containing O6-MeG/T mismatches are as efficient as oligonucleotides containing O6-MeG/C in competing for MGMT repair activity, indicating that O6-MeG mispaired with thymine is still subject to repair by MGMT. The addition of MMR proteins from nuclear extracts, or of recombinant MutSα, to the in vitro repair assay did not affect the repair of O6-MeG/T lesions by MGMT. This indicates that the presence of MutSα still allows access of MGMT to O6-MeG/T lesions. To elucidate the protective effect of MGMT in the first and second replication cycle after N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment, MGMT transfected CHO cells were synchronized and MGMT was inactivated by pulse-treatment with O6-benzylguanine (O6-BG). Thereafter, the recovered cells were treated with MNNG and subjected to clonogenic survival assays. Cells which expressed MGMT in the first and second cell cycle were more resistant than cells which expressed MGMT only in the second (post-treatment) cell cycle. Cells which did not express MGMT in both cell cycles were most sensitive. This indicates that repair of O6-MeG can occur both in the first and second cell cycle after alkylation protecting cells from the killing effect of the lesion.