The human Xrcc3 protein is involved in the repair of damaged DNA through homologous recombination, in which homologous pairing is a key step. The Rad51 protein is believed to be the only protein factor that promotes homologous pairing in recombinational DNA repair in mitotic cells. In the brain, however, Rad51 expression is extremely low, whereas XRCC3, a human homologue of Saccharomyces cerevisiae RAD57 that activates the Rad51-dependent homologous pairing with the yeast Rad55 protein, is expressed. In this study, a two-hybrid analysis conducted with the use of a human brain cDNA library revealed that the major Xrcc3-interacting protein is a Rad51 paralog, Rad51C͞Rad51L2. The purified Xrcc3⅐Rad51C complex, which shows apparent 1:1 stoichiometry, was found to catalyze the homologous pairing. Although the activity is reduced, the Rad51C protein alone also catalyzed homologous pairing, suggesting that Rad51C is a catalytic subunit for homologous pairing. The DNA-binding activity of Xrcc3⅐Rad51C was drastically decreased in the absence of Xrcc3, indicating that Xrcc3 is important for the DNA binding of Xrcc3⅐Rad51C. Electron microscopic observations revealed that Xrcc3⅐Rad51C and Rad51C formed similar filamentous structures with circular single-stranded DNA.

The Xrcc2 and Rad51D/Rad51L3 proteins, which belong to the Rad51 paralogs, are required for homologous recombinational repair (HRR) in vertebrates. The Xrcc2 and Rad51D/Rad51L3 genes, whose products interact with each other, have essential roles in ensuring normal embryonic development. In the present study, we coexpressed the human Xrcc2 and Rad51D/Rad51L3 proteins (Xrcc2 and Rad51D, respectively) in Escherichia coli, and purified the Xrcc2⅐Rad51D complex to homogeneity. The Xrcc2⅐Rad51D complex catalyzed homologous pairing between single-stranded and double-stranded DNA, similar to the function of the Xrcc3⅐ Rad51C complex, which is another complex of the Rad51 paralogs. An electron microscopic analysis showed that Xrcc2⅐Rad51D formed a multimeric ring structure in the absence of DNA. In the presence of ssDNA, Xrcc2⅐Rad-51D formed a filamentous structure, which is commonly observed among the human homologous pairing proteins, Rad51, Rad52, and Xrcc3⅐Rad51C.Chromosomal DNA is vulnerable to attacks from the environment and sustains multiple types of damage, including the double strand break (DSB), 1 which is a lethal DNA lesion for cells if it is not repaired. Homologous recombinational repair (HRR) is one of the major pathways for the repair of DSBs. When cells are defective in HRR, unrepaired DSBs accumulate in chromosomes (1).In Escherichia coli, the RecA protein catalyzes homologous pairing, which is a key step in HRR (2, 3). In homologous pairing, RecA binds the single-stranded DNA (ssDNA) produced at DSB sites and forms nucleoprotein filaments. Then, the nucleoprotein filaments bind double-stranded DNA (dsDNA) and form a three-component complex that includes ssDNA, dsDNA, and RecA. In the three-component complex, the homology between ssDNA and dsDNA is searched, and joint molecules, in which the ssDNA invades into the homologous region of the dsDNA, are formed as products of homologous pairing.In eukaryotes, the Rad51 protein has been identified as a homologue of RecA (4). The Rad51 protein, which is conserved from yeast to human (5), forms a nucleoprotein filament that is strikingly similar to that formed by RecA (6), suggesting their functional similarity in HRR. Actually, the Saccharomyces cerevisiae and human Rad51 proteins (ScRad51 and HsRad51, respectively) catalyze homologous pairing (7-9). We have found that the human Xrcc3 and Rad51C/Rad51L2 proteins (Xrcc3 and Rad51C, respectively) form a complex and catalyze homologous pairing (10) in addition to HsRad51. Both Xrcc3 and Rad51C are members of the Rad51 paralogs (Xrcc2 (11, 12), Xrcc3 (12, 13), Rad51B/hREC2/Rad51L1 (14 -16), Rad51C/ Rad51L2 (17), and Rad51D/Rad51L3 (16,18,19), etc.) and share 20 -30% amino acid identity with HsRad51.The Xrcc2 and Xrcc3 genes were first identified as human genes that complement the DNA damage-sensitive hamster cell lines, irs1 and irs1SF, respectively (13, 20 -23), and both genes were confirmed to be involved in HRR in vivo (24, 25). Cells lacking Xrcc2 or Xrcc3 show extreme sensitivity to DNA cross-linking reagent...

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.