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

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·dT pair should be comparable to the dA·dT pair, the presence of m⁶dG in the template inhibited DNA synthesis. Based on K(m)(app) 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.