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

E T Snow,R S Foote,S Mitra

doi:10.1016/s0021-9258(17)39698-9

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.

Highlights

While these observations are highly suggestive of the miscoding potential ofm'Gua, its true base-pairing capability cannot be determined in the presence of the multiple lesions produced in DNA by alkylating agents (1).To directly measure thebase-pairing kinetics of the alkylated base during in vitro replication, DNA templatescontaining m'Gua as the only modified base were synthesized with terminal deoxynucleotidyltransferase and replicated i n vitro with purified prokaryotic DNA polymerases, namely Escherichia coli DNA polymerase I and T4 and T5 phage DNA polymerases
The results presentedin thispaper support his conclusion. This is in contrast to earlier experimental data, which suggested that only about one-third of the 06-methylguanines mispair with T to give G .C+A
T transitions in vivo and in vitro (7, 9). 0'"ethylguanine paired with thymine about 6 shown) established that the polymerase concentration was limiting to 150times more than with cytosine

Summary

THEJOURNAL OF BIOLOGICALCHEMISTRY

Vol 259, No 13,Issue of July 10,ppP. r8i0n9te5-d8i1n00U, 1S9A84. Base-pairing Properties of 06-Methylguaninein Template DNA during in Vitro DNA Replication*. DNA synthesis using methylated poly(dC-dG) templates leads to incorporation of dTMP only when the template lesions include m'Gua (9). While these observations are highly suggestive of the miscoding potential ofm'Gua, its true base-pairing capability cannot be determined in the presence of the multiple lesions produced in DNA by alkylating agents (1).To directly measure thebase-pairing kinetics of the alkylated base during in vitro replication, DNA templatescontaining m'Gua as the only modified base were synthesized with terminal deoxynucleotidyltransferase and replicated i n vitro with purified prokaryotic DNA polymerases, namely Escherichia coli DNA polymerase I and T4 and T5 phage DNA polymerases. There is substantial indirect evidence that O'-methyl- poration of dT opposite m'dG is a rate-limiting step during guanine pairs with thymine (uracil) bothin vivo and in vitro. replication of the polymer

The kinetics of incorporation of dTMPand dAMP with

Predoctoral fellow supported by National Institutes of Health

DISCUSSION