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 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.
D000818:Animals; D001482:Base Composition; D002417:Cattle; D004247:DNA; D004256:DNA Polymerase I / Q000378:metabolism*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D011071:Poly T; D011089:Polydeoxyribonucleotides; D013604:T-Phages / Q000201:enzymology*; D013698:Templates, Genetic; D013950:Thymus Gland
D011089:Polydeoxyribonucleotides; D011071:Poly T; C017650:polydeoxy(guanine-thymine) nucleotide; D006147:Guanine; D004247:DNA; C008449:O-(6)-methylguanine; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase