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J. Biol. Chem., Vol. 281, Issue 52, 40001-40009, December 29, 2006

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The DNA Binding Preference of RAD52 and RAD59 Proteins

IMPLICATIONS FOR RAD52 AND RAD59 PROTEIN FUNCTION IN HOMOLOGOUS RECOMBINATION^*

From the Sections of Microbiology and of Molecular and Cellular Biology, Center for Genetics and Development, University of California, Davis, California, 95616-8665

We examined the double-stranded DNA (dsDNA) binding preference of the Saccharomyces cerevisiae Rad52 protein and its homologue, the Rad59 protein. In nuclease protection assays both proteins protected an internal sequence and the dsDNA ends equally well. Similarly, using electrophoretic mobility shift assays, we found the affinity of both Rad52 and Rad59 proteins for DNA ends to be comparable with their affinity for internal sequences. The protein-DNA complexes were also directly visualized using atomic force microscopy. Both proteins formed discrete complexes, which were primarily found (90–94%) at internal dsDNA sites. We also measured the DNA end binding behavior of human Rad52 protein and found a slight preference for dsDNA ends. Thus, these proteins have no strong preference for dsDNA ends over internal sites, which is inconsistent with their function at a step of dsDNA break repair that precedes DNA processing. Therefore, we conclude that S. cerevisiae Rad52 and Rad59 proteins and their eukaryotic counterparts function by binding to single-stranded DNA formed as intermediates of recombination rather than by binding to the unprocessed DNA double-strand break.

Received for publication, August 22, 2006 , and in revised form, September 29, 2006.

^* This work was supported by National Institutes of Health Grant GM-62653. 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.

¹ Current address: Dept. of Biological Sciences, 211 Life Sciences Research Facility, Ohio University, Athens, OH 45701.

² To whom correspondence should be addressed: Section of Microbiology, Center for Genetics and Development, One Shields Ave., University of California, Davis, CA 95616-8665. Tel.: 530-752-5938; Fax: 530-752-5939; E-mail: sckowalczykowski{at}ucdavis.edu.

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