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J. Biol. Chem., Vol. 281, Issue 9, 6000-6009, March 3, 2006

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Inhibition of Werner Syndrome Helicase Activity by Benzo[a]pyrene Diol Epoxide Adducts Can Be Overcome by Replication Protein A^*

Saba Choudhary¹, Kevin M. Doherty¹, Christopher J. Handy, Jane M. Sayer, Haruhiko Yagi, Donald M. Jerina, and Robert M. Brosh, Jr.²

From the Laboratory of Molecular Gerontology, NIA, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland 21224 and the Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892

RecQ helicases are believed to function in repairing replication forks stalled by DNA damage and may also play a role in the intra-S-phase checkpoint, which delays the replication of damaged DNA, thus permitting repair to occur. Since little is known regarding the effects of DNA damage on RecQ helicases, and because the replication and recombination defects in Werner syndrome cells may reflect abnormal processing of damaged DNA associated with the replication fork, we examined the effects of specific bulky, covalent adducts at N⁶ of deoxyadenosine (dA) or N² of deoxyguanosine (dG) on Werner (WRN) syndrome helicase activity. The adducts are derived from the optically active 7,8-diol 9,10-epoxide (DE) metabolites of the carcinogen benzo[a]pyrene (BaP). The results demonstrate that WRN helicase activity is inhibited in a strand-specific manner by BaP DE-dG adducts only when on the translocating strand. These adducts either occupy the minor groove without significant perturbation of DNA structure (trans adducts) or cause base displacement at the adduct site (cis adducts). In contrast, helicase activity is only mildly affected by intercalating BaP DE-dA adducts that locally perturb DNA double helical structure. This differs from our previous observation that intercalating dA adducts derived from benzo[c]phenanthrene (BcPh) DEs inhibit WRN activity in a strand- and stereospecific manner. Partial unwinding of the DNA helix at BaP DE-dA adduct sites may make such adducted DNAs more susceptible to the action of helicase than DNA containing the corresponding BcPh DE-dA adducts, which cause little or no destabilization of duplex DNA. The single-stranded DNA binding protein RPA, an auxiliary factor for WRN helicase, enabled the DNA unwinding enzyme to overcome inhibition by either the trans-R or cis-R BaP DE-dG adduct, suggesting that WRN and RPA may function together to unwind duplex DNA harboring specific covalent adducts that otherwise block WRN helicase acting alone.

Received for publication, September 14, 2005 , and in revised form, December 23, 2005.

^* This research was supported by the Intramural Research Program of the NIA and NIDDK (National Institutes of Health). 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 supplemental Figs. 1 and 2.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 410-558-8578; Fax: 410-558-8157; E-mail:broshr{at}grc.nia.nih.gov.

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