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Inhibition of Human O⁶-Methylguanine-DNA Methyltransferase by 5-Methylcytosine

Department of Pharmacology and Toxicology and the Norris Cotton Cancer Center, Dartmouth Medical School, Hanover, New Hampshire 03755-3835

The ability of cloned human O⁶-methylguanine-DNA methyltransferase to repair a methylated guanine in a CpG-containing sequence, i.e., island, was studied by using a synthetic double-stranded 20-mer oligonucleotide from codon 248 of the p53 gene and another designed sequence. The double-stranded oligonucleotides incorporating 5-methylcytosine (5mC) and O⁶-methylguanine (O⁶mG) in various combinations in a CpG site were 5' labeled with ³²P and incubated with recombinant O⁶-methylguanine-DNA methyltransferase. The rate constant for O⁶-methylguanine-DNA methyltransferase repair of O⁶mG in this oligomer was always higher with the substrate which contained only the O⁶mG, as compared to the oligomer that included a 5mC adjacent in the 5'-position to the methylated guanine. The reduction in substrate activity ranged from 75% (modified p53 sequence) to 100% (in the designed oligomer). A 5mC opposite the O⁶mG reduced the rate slightly. These results suggest that O⁶-methylation of the guanine moiety at CpG islands may not be efficiently repaired when normal 5mC is present and this may contribute significantly to an increase in mutagenesis of p53 and like molecules.

¹ Supported as a postdoctoral fellow under Training Grant CA 09658 from the National Cancer Institute.

² Supported in part by Grant CA 36679 from the National Cancer Institute. To whom requests for reprints should be addressed.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5/ 5/93. Accepted 11/26/93.

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