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.