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J. Biol. Chem., Vol. 281, Issue 35, 25205-25214, September 1, 2006

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Domain Structure and DNA Binding Regions of Protein from Bacteriophage ^*

Zengru Wu¹, Xu Xing¹, Casey E. Bohl, James W. Wisler, James T. Dalton², and Charles E. Bell³

From the Division of Pharmaceutics, College of Pharmacy, and Department of Molecular and Cellular Biochemistry, College of Medicine, Ohio State University, Columbus, Ohio 43210

protein from bacteriophage promotes a single-strand annealing reaction that is central to Red-mediated recombination at double-strand DNA breaks and chromosomal ends. protein binds most tightly to an intermediate of annealing formed by the sequential addition of two complementary oligonucleotides. Here we have characterized the domain structure of protein in the presence and absence of DNA using limited proteolysis. Residues 1–130 form an N-terminal "core" domain that is resistant to proteases in the absence of DNA, residues 131–177 form a central region with enhanced resistance to proteases upon DNA complex formation, and the C-terminal residues 178–261 of protein are sensitive to proteases in both the presence and absence of DNA. We probed the DNA binding regions of protein further using biotinylation of lysine residues and mass spectrometry. Several lysine residues within the first 177 residues of protein are protected from biotinylation in the DNA complex, whereas none of the lysine residues in the C-terminal portion are protected. The results lead to a model for the domain structure and DNA binding of protein in which a stable N-terminal core and a more flexible central domain come together to bind DNA, whereas a C-terminal tail remains disordered. A fragment consisting of residues 1–177 of protein maintains normal binding to sequentially added complementary oligonucleotides and has significantly enhanced binding to single-strand DNA.

Received for publication, November 21, 2005 , and in revised form, June 28, 2006.

^* 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. S1–S3.

¹ These authors contributed equally to this work.

² To whom correspondence may be addressed: Division of Pharmaceutics, College of Pharmacy, The Ohio State University, 500 West 12th Ave., Columbus OH 43210. Tel.: 614-688-3797; Fax: 614-292-7766; E-Mail: dalton.1{at}osu.edu. ³ Supported by National Institutes of Health Grant GM067947. To whom correspondence may be addressed: Dept. of Molecular and Cellular Biochemistry, The Ohio State University, 1645 Neil Ave., Columbus, OH 43210. Tel.: 614-688-3115; Fax: 614-292-4118; E-mail: bell.489{at}osu.edu.

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