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J. Biol. Chem., Vol. 279, Issue 6, 4250-4259, February 6, 2004

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Characterization of a Mutagenic DNA Adduct Formed from 1,2-Dibromoethane by O⁶-Alkylguanine-DNA Alkyltransferase^*

Liping Liu, David L. Hachey¶, Gerardo Valadez¶, Kevin M. Williams¶||, F. Peter Guengerich¶, Natalia A. Loktionova, Sreenivas Kanugula, and Anthony E. Pegg**

From the Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and the ^¶Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

It has been proposed that the DNA repair protein O⁶-alkylguanine-DNA alkyltransferase increases the mutagenicity of 1,2-dibromoethane by reacting with it at its cysteine acceptor site to form a highly reactive half-mustard, which can then react with DNA (Liu, L., Pegg, A. E., Williams, K. M., and Guengerich, F. P. (2002) J. Biol. Chem. 277, 37920-37928). Incubation of Escherichia coli-expressed human alkyltransferase with 1,2-dibromoethane and single-stranded oligodeoxyribonucleotides led to the formation of covalent transferaseoligo complexes. The order of reaction determined was Gua>Thy>Cyt>Ade. Mass spectrometry analysis of the tryptic digest of the reaction product indicated that some of the adducts led to depurination with the release of the Gly¹³⁶-Arg¹⁴⁷ peptide cross-linked to a Gua at the N⁷ position, with the site of reaction being the active site Cys¹⁴⁵ as established by chromatographic retention time and the fragmentation pattern determined by tandem mass spectrometry of a synthetic peptide adduct. The alkyltransferase-mediated mutations produced by 1,2-dibromoethane were predominantly Gua to Ade transitions but, in the spectrum of such rifampicin-resistant mutations in the RpoB gene, 20% were Gua to Thy transversions. The latter are likely to have arisen from the apurinic site generated from the Gua-N⁷ adduct. Support exists for an additional adduct/mutagenic pathway because evidence was obtained for DNA adducts other than at the Gua N⁷ atom and for mutations other than those attributable to depurination. Thus, chemical and biological evidence supports the existence of at least two alkyltransferase-dependent pathways for 1,2-dibromoethane-induced mutagenicity, one involving Gua N⁷-alkylation by alkyltransferase-S-CH₂CH₂Br and depurination, plus another as yet uncharacterized system(s).

Received for publication, October 9, 2003 , and in revised form, November 20, 2003.

^* This work was supported in part by United States Public Health Service (USPHS) Grants R01 CA18137 (to A. E. P.) and 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.

The on-line version of this article (available at http://www.jbc.org) contains Supplementary Data.

Present address: Abramson Family Cancer Research Institute, University of Pennsylvania, 421 Curie Blvd., 438 BRBII/III, Philadelphia, Pennsylvania 19104.

^|| Supported in part by USPHS T32 ES07028. Present address: Department of Chemistry, Western Kentucky University, Thompson Complex North Wing 329, 1 Big Red Way, Bowling Green, KY 42101.

^** To whom correspondence should be addressed: Dept. of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, PA 17033. Tel.: 717-531-8152; Fax: 717-531-5157; E-mail: apegg{at}psu.edu.

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