Base-pairing properties of O6-methylguanine in template DNA during in vitro DNA replication.

ABSTRACT

The kinetics of incorporation of deoxynucleotide precursors directed by the promutagenic base, O6-methylguanine (m6Gua), was analyzed during in vitro replication of m6Gua-containing synthetic polydeoxynucleotides by T4 and T5 phage DNA polymerases and Escherichia coli DNA polymerase I. When poly(dT,m6dG) and poly(dC,m6dG) with covalently attached primers were replicated, O6-methylguanine paired with either thymine or cytosine but with a much higher preference for thymine. dCTP and dTTP acted as competitive inhibitors of each other during DNA synthesis. O6-Methylguanine also directed incorporation of dAMP by T5 DNA polymerase. This dAMP incorporation was not inhibited by dTTP. Contrary to theoretical predictions that the m6dG X dT pair should be comparable to the dA X dT pair, the presence of m6dG in the template inhibited DNA synthesis. Based on Kappm values, E. coli DNA polymerase I showed a much higher preference for dTMP incorporation over dCMP opposite m6dG in the template than T4 and T5 DNA polymerases. At the same time, there was a higher turnover of dCTP than of dTTP by the E. coli enzyme. However, in all cases, the turnover of deoxynucleotides during replication of m6Gua-containing templates was more than that observed with templates without the alkylated base.