The human Dmc1 protein, a RecA/Rad51 homolog, is a meiosis-specific DNA recombinase that catalyzes homologous pairing. RecA and Rad51 form helical filaments, while Dmc1 forms an octameric ring. In the present study, we crystallized the full-length human Dmc1 protein and solved the structure of the Dmc1 octameric ring. The monomeric structure of the Dmc1 protein closely resembled those of the human and archaeal Rad51 proteins. In addition to the polymerization motif that was previously identified in the Rad51 proteins, we found another hydrogen bonding interaction at the polymer interface, which could explain why Dmc1 forms stable octameric rings instead of helical filaments. Mutagenesis studies identified the inner and outer basic patches that are important for homologous pairing. The inner patch binds both single-stranded and double-stranded DNAs, while the outer one binds single-stranded DNA. Based on these results, we propose a model for the interaction of the Dmc1 rings with DNA.
In Saccharomyces cerevisiae, the Hop2 protein forms a complex with the Mnd1 protein and is required for the alignment of homologous chromosomes during meiosis, probably through extensive homology matching between them. The Rad51 and Dmc1 proteins, the eukaryotic RecA orthologs, promote strand exchange and may function in the extensive matching of homology within paired DNA molecules. In the present study, we purified the human TBPIP/ Hop2-Mnd1 complex and found that it significantly stimulates the Dmc1-and Rad51-mediated strand exchange. The human Hop2-Mnd1 complex preferentially binds to a three-stranded DNA branch, which mimics the strand-exchange intermediate. These findings are consistent with genetic data, which showed that the Hop2 and Mnd1 proteins are required for homology matching between homologous chromosomes. Therefore, the human TBPIP/ Hop2-Mnd1 complex may ensure proper pairing between homologous chromosomes through its stimulation of strand exchange during meiosis. In meiosis, a high level of homologous recombination occurs only between homologous chromosomes but not between sister chromatids. This meiotic homologous recombination is initiated by the formation of a double strand break (DSB), 3 which is introduced by the SPO11 protein (1-3). On the other hand, in mitosis, homologous recombination functions to repair DSBs, which are introduced by DNA damaging agents, such as ionizing radiation, DNA cross-linking reagents, oxidative stress, and replication errors. This mitotic homologous recombination repair mainly occurs between sister chromatids or between the abundant intra-and inter-chromosomal homologous repeat sequences.After the DSB formation, a single-stranded DNA (ssDNA) tail derived from a DSB site invades the homologous double-stranded DNA (dsDNA). This strand-invasion step, called homologous pairing, primes heteroduplex formation, in which new Watson-Crick base pairs are formed between the invading strand and its complementary strand of parental dsDNA. Then, the heteroduplex region is expanded by the subsequent strand-exchange step. This strand-exchange step may be important for the extensive matching of homology between paired chromosomes to ensure that homologous recombination occurs between the proper partners. The Escherichia coli RecA protein is known to catalyze the homologous pairing and strand-exchange steps (4 -7), and two RecA homologues, the Rad51 and Dmc1 proteins, have been identified in eukaryotes (8 -11). The Rad51 and Dmc1 proteins have been shown to catalyze strand exchange in vitro, but their strandexchange activities are low as compared with that of .Genetic studies with Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Arabidopsis thaliana have identified the HOP2, meu13 ϩ , and AHP2 genes, respectively, as being essential for meiotic homologous recombination (15-17). The HOP2, meu13 ϩ , and AHP2 genes are orthologs, and the human and mouse TBPIP proteins have been identified as mammalian orthologs of Hop2 (18,19). The hop2 deletion mutant in S. cerevisiae fails...
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 single-stranded DNA (ssDNA) binding protein from Escherichia coli (EcoSSB) plays a central role in DNA replication, recombination and repair. The tertiary structure of EcoSSB was determined at 2.2 A resolution. This is rather higher resolution than previously reported. Crystals were grown from the homogeneous intact protein but the EcoSSB tetramer in the crystals contains truncated subunits lacking a part of the C-terminal. The structure determined includes biologically important flexible loops and C-terminal regions, and revealed the existence of concavities. These concavities include the residues important for ssDNA binding. An ssDNA can be fitted on the concavities and further stabilized through interactions with the loops forming flexible lids. It seems likely to play a central role in the binding of ssDNA.
The RAD52 epistasis group genes are involved in homologous recombination, and they are conserved from yeast to humans. We have cloned a novel human gene, RAD54B, which is homologous to yeast and human RAD54. Human Rad54B (hRad54B) shares high homology with human Rad54 (hRad54) in the central region containing the helicase motifs characteristic of the SNF2/SWI2 family of proteins, but the N-terminal domain is less conserved. In yeast, another RAD54 homolog, TID1/RDH54, plays a role in recombination. Tid1/Rdh54 interacts with yeast Rad51 and a meiosis-specific Rad51 homolog, Dmc1. The N-terminal domain of hRad54B shares homology with that of Tid1/Rdh54, suggesting that Rad54B may be the human counterpart of Tid1/Rdh54. We purified the hRad54 and hRad54B proteins from baculovirus-infected insect cells and examined their biochemical properties. hRad54B, like hRad54, is a DNA-binding protein and hydrolyzes ATP in the presence of double-stranded DNA, though its rate of ATP hydrolysis is lower than that of hRad54. Human Rad51 interacts with hRad54 and enhances its ATPase activity. In contrast, neither human Rad51 nor Dmc1 directly interacts with hRad54B. Although hRad54B is the putative counterpart of Tid1/Rdh54, our findings suggest that hRad54B behaves differently from Tid1/Rdh54.
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