Repair of O-6-methylguanine is not affected by thymine base pairing and the presence of MMR proteins

Methylation at the O-6-position of guanine (O-6-MeG) by alkylating agents i s efficiently removed by O-6-Methylguanine-DNA methyltransferase (MGMT), pr eventing from cytotoxic, mutagenic, clastogenic and carcinogenic effects Of O-6 O-6-MeG-inducing agents. If O-6-MeG is not removed from DNA prior to r eplication, thymine will be incorporated instead of cytosine opposite the O -6-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 O-6-MeG mispaired with th ymine (O-6-MeG/T)? (2) Do MMR proteins interfere with the repair of O-6-MeG /T by MGMT? (3) Does MGMT show a protective effect if it is expressed after replication of -MeG/T mismatches are as DNA containing O-6-MeG? Using an i n vitro assay we show that oligonucleotides containing O-6 efficient as oli gonucleotides containing O-6-MeG/C in competing for MGMT repair activity, i ndicating that O-6-MeG mispaired with thymine is still subject to repair by MGMT. The addition of MMR proteins from nuclear extracts, or of recombinan t MutS alpha, to the in vitro repair assay did not affect the repair of O-6 -MeG/T lesions by MGMT. This indicates that the presence of MutS alpha stil l allows access of MGMT to O-6-MeG/T lesions. To elucidate the protective e ffect of MGMT in the first and second replication cycle after N-methyl-N'-n itro-N-nitrosoguanidine (MNNG) treatment, MGMT transfected CHO cells were s ynchronized and MGMT was inactivated by pulse-treatment with O-6-benzylguan ine (O-6-BG). Thereafter, the recovered cells were treated with MNNG and su bjected to clonogenic survival assays. Cells which expressed MGMT in the fi rst and second cell cycle were more resistant than cells which expressed MG MT only in the second (post-treatment) cell cycle. Cells which did not expr ess MGMT in both cell cycles were most sensitive. This indicates that repai r of O-6-MeG can occur both in the first and second cell cycle after alkyla tion protecting cells from the killing effect of the lesion. (C) 2001 Elsev ier Science B.V. Ail rights reserved.