Cell lines deficient in certain genes of the DNA mismatch repair (MMR) system show a marked resistance to the cell killing or apoptotic effects of DNA alkylating agents. The molecular mechanism for this resistance is not yet understood, but is currently of great interest because of the recent use of these agents in cancer chemotherapy. Of particular interest is the role of the primary mutagenic base-adduct, 06-methylguanine (06-meG), in this MMR-dependent resistance. In this paper, an assay for testing one of the hypotheses for the MMR-dependent resistance to alkylating agents (the futile repair cycle) is described and the progress towards performing such an assay is reported. Specifically, single-stranded phage DNA was prepared with less than 5% double-stranded DNA contaminant, fl phage gene II-protein was purified with a specific activity of approximately 5% of the published value using an abbreviated protocol, and MMRcompetent HeLa cell nuclear extract was prepared with an in vitro repair efficiency of 30%. Also, the possibility of improving in vitro repair assays by using alkaline phosphatase in DNA substrate preparation is investigated.
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