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J. Biol. Chem., Vol. 279, Issue 43, 45245-45256, October 22, 2004

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Analyzing the Handoff of DNA from UvrA to UvrB Utilizing DNA-Protein Photoaffinity Labeling^*

Matthew J. DellaVecchia, Deborah L. Croteau, Milan Skorvaga¶, Sergey V. Dezhurov||**, Olga I. Lavrik||**, and Bennett Van Houten

From the Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, the ^¶Department of Molecular Genetics, Cancer Research Institute, Slovak Academy of Sciences, Vlarska 7, 833 91 Bratislava, Slovakia, and the ^||Novosibirsk Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, Lavrentena, 8, Novosibirsk 630090, Russia

To better define the molecular architecture of nucleotide excision repair intermediates it is necessary to identify the specific domains of UvrA, UvrB, and UvrC that are in close proximity to DNA damage during the repair process. One key step of nucleotide excision repair that is poorly understood is the transfer of damaged DNA from UvrA to UvrB, prior to incision by UvrC. To study this transfer, we have utilized two types of arylazido-modified photoaffinity reagents that probe residues in the Uvr proteins that are closest to either the damaged or non-damaged strands. The damaged strand probes consisted of dNTP analogs linked to a terminal arylazido moiety. These analogs were incorporated into double-stranded DNA using DNA polymerase and functioned as both the damage site and the cross-linking reagent. The non-damaged strand probe contained an arylazido moiety coupled to a phosphorothioate-modified backbone of an oligonucleotide opposite the damaged strand, which contained an internal fluorescein adduct. Six site-directed mutants of Bacillus caldotenax UvrB located in different domains within the protein (Y96A, E99A, R123A, R183E, F249A, and D510A), and two domain deletions (2 and -hairpin), were assayed. Data gleaned from these mutants suggest that the handoff of damaged DNA from UvrA to UvrB proceeds in a three-step process: 1) UvrA and UvrB bind to the damaged site, with UvrA in direct contact; 2) a transfer reaction with UvrB contacting mostly the non-damaged DNA strand; 3) lesion engagement by the damage recognition pocket of UvrB with concomitant release of UvrA.

Received for publication, July 29, 2004 , and in revised form, August 11, 2004.

^* 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.

This article was selected as a Paper of the Week.

These authors contributed equally to the manuscript.

^** These authors were supported in part by Human Frontier Science Program Grant RGP0007/2004-C104 and by Russian Foundation for Basic Research Grant 03-04-48562. 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.

To whom correspondence should be addressed. Tel.: 919-541-2799; Fax: 919-541-7593; E-mail: vanhout1{at}niehs.nih.gov.

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