Nucleic Acids Research, 2003, Vol. 31, No. 14 4041-4050
© 2003 Oxford University Press
Region and amino acid residues required for Rad51C binding in the human Xrcc3 protein
1 RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan, 2 Cellular Signaling Laboratory, RIKEN Harima Institute at SPring-8, 1-1-1 Kohto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan, 3 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, 1637 Yana, Kisarazu, Chiba 292-0812, Japan, 4 Department of Electrical Engineering and Bioscience, School of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan, 5 Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan and 6 Cellular and Molecular Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
*To whom correspondence should be addressed. Tel: +81 45 503 9196; Fax: +81 45 503 9201; Email: tshibata{at}postman.riken.go.jp
Correspondence may also be addressed to S. Yokoyama. Tel: +81 48 467 9537; Fax: +81 48 462 4671; Email: yokoyama{at}biochem.s.u-tokyo.ac.jp
The Xrcc3 protein, which is required for the homologous recombinational repair of damaged DNA, forms a complex with the Rad51C protein in human cells. Mutations in either the Xrcc3 or Rad51C gene cause extreme sensitivity to DNA-damaging agents and generate the genomic instability frequently found in tumors. In the present study, we found that the Xrcc3 segment containing amino acid residues 63–346, Xrcc363–346, is the Rad51C-binding region. Biochemical analyses revealed that Xrcc363–346 forms a complex with Rad51C, and the Xrcc363–346– Rad51C complex possesses ssDNA and dsDNA binding abilities comparable to those of the full-length Xrcc3–Rad51C complex. Based on the structure of RecA, which is thought to be the ancestor of Xrcc3, six Xrcc3 point mutants were designed. Two-hybrid and biochemical analyses of the Xrcc3 point mutants revealed that Tyr139 and Phe249 are essential amino acid residues for Rad51C binding. Superposition of the Xrcc3 Tyr139 and Phe249 residues on the RecA structure suggested that Tyr139 may function to ensure proper folding and Phe249 may be important to constitute the Rad51C-binding interface in Xrcc3.
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