Repair of o-propylguanine and o-butylguanine in dna by o-alkylguanine-dna alkyltransferases from rat liver and E. coli

DNA substrates containing O⁶-n-butylguanine, O⁶-iso-butylguanine, O⁶-n-propylguanine and O⁶-iso-propylguanine were prepared by reaction of calf thymus DNA with the appropriate N-alkyl-N-nitrosourea. These substrates were used to test the ability of O⁶-alkylguanine-DNA alkyltransferases from Escherichia coli and rat liver to remove such alkyl groups from the O⁶-position of guanine. It was found that all of these adducts were removed by the alkyltransferases, but the branched alkyl chain iso-butyl- and iso-propyl adducts were removed very slowly. Also, when tested with a DNA substrate containing both O⁶-n-propylguanine and O⁶-iso-propylguanine, the alkyltransferases removed almost all of the n-propyl-adduct before the iso-propyl-adduct was attacked. Both alkyltransferases showed a decreasing rate of reaction as the size of the alkyl group increased, but there was a significant difference between the rat liver and E. coli alkyltransferase in the relative rates. The rat liver alkyltransferase repaired O⁶-methylguanine more slowly than the E. coli protein, but was considerably more rapid than the bacterial equivalent when acting on n-propyl- and n-butyl-adducts. The relative rates of repair were methyl > ethyl > n-propyl > n-butyl >iso-propyl, iso-butyl for the E. coli alkyltransferase and methyl > ethyl, n-propyl > n-butyl > iso-propyl, iso-butyl > 2-hydroxyethyl for the rat liver protein. These results indicate that differential rates of repair may contribute to the relative risks of carcinogenesis and mutagenesis by exposure to alkylating agents of different size and that rates of repair may be species specific and must be determined from specific measurements rather than extrapolated from data on other organisms.