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

E. T. Snow, R. S. Foote, S. Mitra

Research output: Contribution to journalArticle

133 Scopus citations

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·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.

Original languageEnglish (US)
Pages (from-to)8095-8100
Number of pages6
JournalJournal of Biological Chemistry
Volume259
Issue number13
StatePublished - 1984

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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