title: Subunit interface residues F129 and H294 of human RAD51 are essential for recombinase function.
abstract: RAD51 mediated homologous recombinational repair (HRR) of DNA double-strand breaks (DSBs) is essential to maintain genomic integrity. RAD51 forms a nucleoprotein filament (NPF) that catalyzes the fundamental homologous pairing and strand exchange reaction (recombinase) required for HRR. Based on structural and functional homology with archaeal and yeast RAD51, we have identified the human RAD51 (HsRAD51) subunit interface residues HsRad51(F129) in the Walker A box and HsRad51(H294) in the L2 ssDNA binding region as potentially important participants in salt-induced conformational transitions essential for recombinase activity. We demonstrate that the HsRad51(F129V) and HsRad51(H294V) substitution mutations reduce DNA dependent ATPase activity and are largely defective in the formation of a functional NPF, which ultimately eliminates recombinase catalytic functions. Our data are consistent with the conclusion that the HsRAD51(F129) and HsRAD51(H294) residues are important participants in the cation-induced allosteric activation of HsRAD51.
authors.affiliation: Biophysics Graduate Program, The Ohio State University Medical Center, Columbus, Ohio, United States of America.
authors.forename: Ravindra
authors.initials: R
authors.lastname: Amunugama
authors.forename: Richard
authors.initials: R
authors.lastname: Fishel
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051135:Rad51 Recombinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D059767:Recombinational DNA Repair; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance
chemical_list: D004277:DNA, Single-Stranded; D021122:Protein Subunits; D010649:Phenylalanine; D006639:Histidine; D000255:Adenosine Triphosphate; D004247:DNA; C495153:RAD51 protein, human; D051135:Rad51 Recombinase