The Rad51 proteins are the eukaryotic orthologs of the bacterial RecA protein [1], which promotes key steps in homologous recombination [2][3][4][5]. A RAD51 null mutation causes severe defects in meiotic homologous recombination and mitotic recombinational repair of double strand breaks (DSBs) in Saccharomyces cerevisiae [1]. Rad51 is thus required for both the meiotic and mitotic homologous recombination processes, while another ortholog, Dmc1, is specific to meiotic homologous recombination [6][7][8]. In higher eukaryotes, Rad51 is even essential for cell survival: disruption of the RAD51 gene in mice results in early embryonic lethality [9,10] and the RAD51 gene knockout in chicken DT40 cells causes cell death, with the accumulation of spontaneous chromosomal breaks [11].Rad51 and RecA apparently use similar mechanisms to promote homologous recombination [12][13][14][15]. During the homologous recombination process, Rad51 is thought to bind single-stranded tails produced at DSB sites, and to form a helical nucleoprotein filament. The single-stranded DNA (ssDNA) and double-stranded The human Rad51 protein, a eukaryotic ortholog of the bacterial RecA protein, is a key enzyme that functions in homologous recombination and recombinational repair of double strand breaks. The Rad51 protein contains two flexible loops, L1 and L2, which are proposed to be sites for DNA binding, based on a structural comparison with RecA. In the present study, we performed mutational and fluorescent spectroscopic analyses on the L1 and L2 loops to examine their role in DNA binding. Gel retardation and DNA-dependent ATP hydrolysis measurements revealed that the substitution of the tyrosine residue at position 232 (Tyr232) within the L1 loop with alanine, a short side chain amino acid, significantly decreased the DNA-binding ability of human Rad51, without affecting the protein folding or the salt-induced, DNA-independent ATP hydrolysis. Even the conservative replacement with tryptophan affected the DNA binding, indicating that Tyr232 is involved in DNA binding. The importance of the L1 loop was confirmed by the fluorescence change of a tryptophan residue, replacing the Asp231, Ser233, or Gly236 residue, upon DNA binding. The alanine replacement of phenylalanine at position 279 (Phe279) within the L2 loop did not affect the DNA-binding ability of human Rad51, unlike the Phe203 mutation of the RecA L2 loop. The Phe279 side chain may not be directly involved in the interaction with DNA. However, the fluorescence intensity of the tryptophan replacing the Rad51-Phe279 residue was strongly reduced upon DNA binding, indicating that the L2 loop is also close to the DNA-binding site.Abbreviations DSB, double strand break; dsDNA, double-stranded DNA; HsRad51, Homo sapiens Rad51; RPA, replication protein A; ScRad51, Saccharomyces cerevisiae Rad51; ssDNA, single-stranded DNA; SSB, single stranded DNA-binding protein.

The M200V polymorphism of the human DMC1 protein, which is an essential, meiosis-specific DNA recombinase, was found in an infertile patient, raising the question of whether this homozygous human DMC1-M200V polymorphism may cause infertility by affecting the function of the human DMC1 protein. In the present study, we determined the crystal structure of the human DMC1-M200V variant in the octameric-ring form. Biochemical analyses revealed that the human DMC1-M200V variant had reduced stability, and was moderately defective in catalyzing in vitro recombination reactions. The corresponding M194V mutation introduced in the Schizosaccharomyces pombe dmc1 gene caused a significant decrease in the meiotic homologous recombination frequency. Together, these structural, biochemical and genetic results provide extensive evidence that the human DMC1-M200V mutation impairs its function, supporting the previous interpretation that this single-nucleotide polymorphism is a source of human infertility.