Region and amino acid residues required for Rad51C binding in the human Xrcc3 protein.
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, Xrcc3(63-346), is the Rad51C-binding region. Biochemical analyses revealed that Xrcc3(63-346) forms a complex with Rad51C, and the Xrcc3(63-346)- 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.
Kurumizaka
Hitoshi
H
RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
Enomoto
Rima
R
Nakada
Maki
M
Eda
Keiko
K
Yokoyama
Shigeyuki
S
Shibata
Takehiko
T
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010649:Phenylalanine / Q000235:genetics; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques; D014443:Tyrosine / Q000235:genetics
D000596:Amino Acids; D004268:DNA-Binding Proteins; C448305:RAD51C protein, human; C110901:X-ray repair cross complementing protein 3; D014443:Tyrosine; D010649:Phenylalanine