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J. Biol. Chem., Vol. 280, Issue 28, 26303-26311, July 15, 2005

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Gly-103 in the N-terminal Domain of Saccharomyces cerevisiae Rad51 Protein Is Critical for DNA Binding^*

Xiao-Ping Zhang, Kyung-Im Lee, Jachen A. Solinger, Konstantin Kiianitsa¶, and Wolf-Dietrich Heyer||**

From the Section of Microbiology, ^||Section of Molecular and Cellular Biology, and ^**Center for Genetics and Development, University of California, Davis, California 95616-8665

Rad51 is a homolog of the bacterial RecA protein and is central for recombination in eukaryotes performing homology search and DNA strand exchange. Rad51 and RecA share a core ATPase domain that is structurally similar to the ATPase domains of helicases and the F1 ATPase. Rad51 has an additional N-terminal domain, whereas RecA protein has an additional C-terminal domain. Here we show that glycine 103 in the N-terminal domain of Saccharomyces cerevisiae Rad51 is important for binding to single-stranded and duplex DNA. The Rad51-G103E mutant protein is deficient in DNA strand exchange and ATPase activity due to a primary DNA binding defect. The N-terminal domain of Rad51 is connected to the ATPase core through an extended elbow linker that ensures flexibility of the N-terminal domain. Molecular modeling of the Rad51-G103E mutant protein shows that the negatively charged glutamate residue lies on the surface of the N-terminal domain facing a positively charged patch composed of Arg-260, His-302, and Lys-305 on the ATPase core domain. A possible structural explanation for the DNA binding defect is that a charge interaction between Glu-103 and the positive patch restricts the flexibility of the N-terminal domain. Rad51-G103E was identified in a screen for Rad51 interaction-deficient mutants and was shown to ablate the Rad54 interaction in two-hybrid assays (Krejci, L., Damborsky, J., Thomsen, B., Duno, M., and Bendixen, C. (2001) Mol. Cell. Biol. 21, 966–976). Surprisingly, we found that the physical interaction of Rad51-G103E with Rad54 was not affected. Our data suggest that the two-hybrid interaction defect was an indirect consequence of the DNA binding defect.

Received for publication, March 24, 2005 , and in revised form, May 19, 2005.

^* This work was supported by National Institutes of Health Grant GM58015 (to W.-D. H.) and Susan G. Komen Breast Cancer Foundation Postdoctoral Fellowship PDF403213 (to X.-P. Z.). 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 arccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Chemistry and Biochemistry, San Francisco State University, 1600 Holloway Ave., San Francisco, CA 94132.

^¶ Present address: University of Washington, Division of Medical Genetics, Seattle, WA 98195-7720.

To whom correspondence should be addressed: Section of Microbiology, University of California, Davis, One Shields Ave., Davis, CA 95616-8665. Tel.: 530-752-3001; Fax: 530-752-3011; E-mail: wdheyer{at}ucdavis.edu.

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				W.-D. Heyer, X. Li, M. Rolfsmeier, and X.-P. Zhang Rad54: the Swiss Army knife of homologous recombination? Nucleic Acids Res., September 10, 2006; 34(15): 4115 - 4125. [Abstract] [Full Text] [PDF]