Kinetics of repair of O⁶-methylguanine in DNA by O⁶-methylguanine-DNA methyltransferase in, vitro and in, vivo

The demethylation of O⁶-methylguanine in double stranded DNA catalyzed by rat liver O⁶-methylguanine-DNA transmethylase was found to proceed much more rapidly when the DNA substrate was methylated to a high extent. When the content of O⁶-methylguanine in DNA was equal to 1 in 2000 guanines, the reaction was 90% complete within 2 min, but when the content was 1 in 500,000 it required 27 min at 37°C. These results suggest that the repair protein either moves along the DNA substrate or else has little selectivity for binding specifically to the sites containing O⁶-methylguanine rather than to the normal DNA. The repair of O⁶-methylguanine in rat liver in, vivo occurred at rates comparable to those seen in, vitro with the substrates alkylated to low extents and was virtually complete within 3 hours. These results provide strong evidence that this protein is the factor responsible for O⁶-methylguanine removal in, vivo and explain the wide variation in time courses reported in the literature since substrates methylated to greatly different extents have been used for such experiments.