Mutagenic Properties of O exp 6 -Methylguanine in DNA in Vivo and in Vitro

摘要

Among the many basic alkylated sites produced in DNA by simple alkylating mutagens, O exp 6 -alkylguanine is believed to be the major promutagenic lesion. Using O exp 6 -methyl dGTP as a substrate during in vitro synthesis of biologically active T7 DNA we have shown that the incorporation of the alkylated nucleotide leads to increased reversion of an amber mutation in the phage DNA. We have shown that O exp 6 -methylguanine has a strong preference for pairing with thymine over cytosine during in vitro replication of synthetic DNA templates with T4 and T5 phage DNA polymerases and E. coli DNA polymerase I. The degree of preference depends not only on the DNA polymerase used but possibly on other factors as well, including the divalent metal ion cofactors. The alkylated base in the template DNA also inhibits chain elongation of the product strand. While O exp 6 -methyl dGTP can act as a substrate for DNA polymerases, as an analog of dATP, the alkylated nucleotide is incorporated only infrequently and inhibits overall DNA synthesis at the same time. A much higher turnover of the alkylated nucleotide than that of the normal nucleotides suggests that O exp 6 -methylguanine is not recognized as a normal base by the DNA polymerases in spite of the fact that it is predicted to form a stable base pair with thymine. (ERA citation 09:010764)