Journal Article

Enhanced <i>in vivo</i> repair of <i>O</i><sup>4</sup>-methylthymine by a mutant human DNA alkyltransferase

Lance P. Encell and Lawrence A. Loeb

in Carcinogenesis

Volume 21, issue 7, pages 1397-1402
Published in print July 2000 | ISSN: 0143-3334
Published online July 2000 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/21.7.1397
Enhanced in vivo repair of O4-methylthymine by a mutant human DNA alkyltransferase

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The repair of O6-methylguanine (m6G) by human O6-alkylguanine-DNA alkyltransferase (hAGT) is ~5000-fold greater than that for O4-methylthymine (m4T). To evaluate each adduct's contribution to mutagenesis, we previously created a mutant hAGT with increased specificity for m4T in vitro. The mutant and wild-type (WT) hAGT have now been expressed in bacterial strains that allow for the specific detection of A:T→G:C and G:C→A:T mutations induced by m4T and m6G, respectively. After exposure to the mutagenic methylating agent, N-methyl-N′-nitro-N-nitrosoguanidine, A:T→G:C substitutions were reduced >4-fold in cells expressing the mutant hAGT compared with 1.1-fold for WT hAGT. G:C→A:T substitutions were decreased >2.5-fold in cells expressing the mutant hAGT, whereas WT hAGT totally prevented G:C→A:T mutations. These results demonstrate that the altered substrate specificity of hAGT observed in vitro also occurs in vivo, and that it is responsible for the observed differences in mutations.

Keywords: BG, O6-benzylguanine; hAGT, human O6-alkylguanine-DNA alkyltransferase; m6G, O6-methylguanine; MNNG, N-methyl-N′-nitro-N-nitrosoguanidine; m4T, O4-methylthymine; WT, wild-type.

Journal Article.  4487 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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