Structure of the hydrogen bonding complex of O⁶-methylguanine with cytosine and thymine during DNA replication

During DNA replication, mutations occur when an incorrect dNTP is incorporated opposite a carcinogen-modified nucleotide. We have probed the structures of the interaction between O⁶-methylguanine (O⁶mG) and cytosine and thymine during replication by kinetic means in order to examine the structure during the rate determining step. The kinetics of incorporation of dCTP and dTTP opposite O⁶mG and three analogs, S⁶-methyl-6-thioguanine, O⁶-methyl-1-deazaguanine and O⁶-methylhypoxanthine, have been measured with four polymerases, the Klenow fragment of DNA polymerase 1, the Klenow fragment with the proof-reading exonuclease inactivated, Taq and Tth polymerases. In the insertion of dTTP opposite O⁶mG, a large decrease in V(max)/K(m) was observed only upon modification of the N1 position. This result is consistent with a Watson-Crick type configuration, for the incorporation of dCTP, the V(max)/K(m) was significantly decreased only with removal of the exocyclic amino group at the 2 position. The pH dependence of the ratio of incorporation of dCTP and dTTP was independent of pH at physiological pH. This result suggests that dCTP is incorporated via an uncharged complex such as the wobble configuration.