Homologous Pairing and Ring and Filament Structure Formation Activities of the Human Xrcc2·Rad51D Complex

Hitoshi Kurumizaka, Shukuko Ikawa, Maki Nakada, Rima Enomoto, Wataru Kagawa, Takashi Kinebuchi, Mitsuyoshi Yamazoe, Shigeyuki Yokoyama, Takehiko Shibata
2002 Journal of Biological Chemistry  
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
more » ... x 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 reagents like cisplatin and ionizing radiation and have significantly increased missegregation of chromosomes (12, 13, 26) . Knock-out experiments in the chicken DT40 cell lines showed that the Rad51B/Rad51L1, Rad51C/Rad51L2, and Rad51D/Rad51L3 genes, as well as the Xrcc2 and Xrcc3 genes, are involved in the HRR pathway (27, 28) . The Xrcc2 protein also has homology to the S. cerevisiae Rad55 protein (29), which interacts with the S. cerevisiae Rad57 protein (30 -32). Interestingly, the Xrcc2 knock-out mice exhibited embryonic lethality associated especially with a high frequency of apoptotic death of neurons in the developing brain (33). Xrcc2 interacts with the human Rad51D/Rad51L3 protein (Rad51D), another Rad51 paralog (34, 35). The Rad51D knock-out mice also showed embryonic lethality (36), indicating that both Xrcc2 and Rad51D are essential for embryonic development. In the present study, we purified the Xrcc2⅐Rad51D complex and characterized it biochemically. The purified Xrcc2⅐Rad51D complex catalyzed homologous pairing between ssDNA and dsDNA, similar to that of the Xrcc3⅐Rad51C complex. The Xrcc2⅐Rad51D complex formed a multimeric ring structure in
doi:10.1074/jbc.m105719200 pmid:11834724 fatcat:t5vescmvhng4bksvzwpmsk2fh4