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J. Biol. Chem., Vol. 281, Issue 27, 18549-18559, July 7, 2006

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Identification of the RecR Toprim Domain as the Binding Site for both RecF and RecO

A ROLE OF RecR IN RecFOR ASSEMBLY AT DOUBLE-STRANDED DNA-SINGLE-STRANDED DNA JUNCTIONS^*

Masayoshi Honda, Jin Inoue, Masatoshi Yoshimasu^¶, Yutaka Ito^¶||, Takehiko Shibata^¶**, and Tsutomu Mikawa^¶**1

From the RIKEN Discovery Research Institute, 2-1, Hirosawa, Wako, Saitama 351-0198, Japan, the Graduate School of Integrated Science, Yokohama City University, 1-7-29, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan, the ^¶Core Research for Evolutional Science and Technology/Japan Science and Technology Agency, Kawaguchi, Saitama 560-0082, the ^||Department of Chemistry, Tokyo Metropolitan University, 1-1, Minami-osawa, Hachioji-shi, Tokyo 192-0397, Japan, and the ^**RIKEN Harima Institute/Spring-8, Mikazuki cho, Hyogo 679-5148, Japan

The RecR protein forms complexes with RecF or RecO that direct the specific loading of RecA onto gapped DNA. However, the binding sites of RecF and RecO on RecR have yet to be identified. In this study, a Thermus thermophilus RecR dimer model was constructed by NMR analysis and homology modeling. NMR titration analysis suggested that the hairpin region of the helix-hairpin-helix motif in the cavity of the RecR dimer is a binding site for double-stranded DNA (dsDNA) and that the acidic cluster region of the Toprim domain is a RecO binding site. Mutations of Glu-84, Asp-88, and Glu-144 residues comprising that acidic cluster were generated. The E144A and E84A mutations decreased the binding affinity for RecO, but the D88A did not. Interestingly, the binding ability to RecF was abolished by E144A, suggesting that the region surrounding the RecR Glu-144 residue could be a binding site not only for RecO but also for RecF. Furthermore, RecR and RecF formed a 4:2 heterohexamer in solution that was unaffected by adding RecO, indicating a preference by RecR for RecF over RecO. The RecFR complex is considered to be involved in the recognition of the dsDNA-ssDNA junction, whereas RecO binds single-stranded DNA (ssDNA) and ssDNA-binding protein. Thus, the RecR Toprim domain may contribute to the RecO interaction with RecFR complexes at the dsDNA-ssDNA junction site during recombinational DNA repair mediated by the RecFOR.

Received for publication, November 28, 2005 , and in revised form, April 6, 2006.

^* This work was supported in part by grants-in-aid from the Japan Society for the Promotion of Science and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology (JST) (to T. S., Y. I., and T. M.). 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 To whom correspondence should be addressed: Bio-supramolecular Structure-Function Group, RIKEN Discovery Research Inst., 1-7-29, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan. Tel.: 81-45-508-7224; Fax: 81-45-508-7364; E-mail: mikawa{at}riken.jp.

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