The essential functions of human Rad51 are independent of ATP hydrolysis

Genetic recombination and the repair of double-strand DNA breaks in Sacchar omyces cerevisiae require Rad51, a homologue of the Escherichia coli RecA p rotein. In vitro, Rad51 binds DNA to form an extended nucleoprotein filamen t and catalyzes the ATP-dependent exchange of DNA between molecules with ho mologous sequences. Vertebrate Rad51 is essential for cell proliferation. U sing site-directed mutagenesis of highly conserved residues of human Rad51 (hRad51) and gene targeting of the RAD51 locus in chicken DT40 cells, we ex amined the importance of Rad51's highly conserved ATP-binding domain. Mutan t hRad51 incapable of ATP hydrolysis (hRad51K-133R) binds DNA less efficien tly than the wild type but catalyzes strand exchange between homologous DNA s. hRad51 does not need to hydrolyze ATP to allow vertebrate cell prolifera tion, form nuclear foci, or repair radiation-induced DNA damage. However, c ells expressing hRad51K-133R show greatly reduced targeted integration freq uencies. These findings show that ATP hydrolysis is involved in DNA binding by hRad51 and suggest that the extent of DNA complexed with hRad51 in nucl eoprotein influences the efficiency of recombination.