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J. Biol. Chem., Vol. 279, Issue 14, 13435-13446, April 2, 2004

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S-(2-Chloroethyl)glutathione-generated p53 Mutation Spectra Are Influenced by Differential Repair Rates More than Sites of Initial DNA Damage^*

J. Gerardo Valadez¶ and F. Peter Guengerich||

From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 and Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del IPN, México DF 07360, Mexico

Several steps occur between the reaction of a chemical with DNA and a mutation, and each may influence the resulting mutation spectrum, i.e. nucleotides at which the mutations occur. The half-mustard S-(2-bro-moethyl)glutathione is the reactive conjugate implicated in ethylene dibromide-induced mutagenesis attributed to the glutathione-dependent pathway. A human p53-driven Ade reporter system in yeast was used to study the factors involved in producing mutations. The synthetic analog S-(2-chloroethyl)glutathione was used to produce DNA damage; the damage to the p53 exons was analyzed using a new fluorescence-based modification of ligation-mediated polymerase chain reaction and an automated sequencer. The mutation spectrum was strongly dominated by the G to A transition mutations seen in other organisms with S-(2-chloroethyl)glutathione or ethylene dibromide. The mutation spectrum clearly differed from the spontaneous spectrum or that derived from N-ethyl,N-nitrosourea. Distinct differences were seen between patterns of modification of p53 DNA exposed to the mutagen in vitro versus in vivo. In the four p53 exons in which mutants were analyzed, the major sites of mutation matched the sites with long half-lives of repair much better than the sites of initial damage. However, not all slowly repaired sites yielded mutations in part because of the lack of effect of mutations on phenotype. We conclude that the rate of DNA repair at individual nucleotides is a major factor in influencing the mutation spectra in this system. The results are consistent with a role of N⁷-guanyl adducts in mutagenesis.

Received for publication, November 11, 2003 , and in revised form, January 5, 2004.

^* This work was supported in part by United States Public Health Service Grants R01 ES10546 and P30 ES00267 (to F. P. G.). 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.

^¶ Present address: Dept. of Neurology, Vanderbilt University School of Medicine, Nashville, TN 37232.

^|| To whom correspondence should be addressed: Dept. of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Robinson Research Bldg., 23rd and Pierce Aves., Nashville, TN 37232-0146. Tel.: 615-322-2261; Fax: 615-322-3141; E-mail: f.guengerich{at}vanderbilt.edu.

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