Insights into the mechanism of Rad51 recombinase from the structure and properties of a filament interface mutant.
Rad51 protein promotes homologous recombination in eukaryotes. Recombination activities are activated by Rad51 filament assembly on ssDNA. Previous studies of yeast Rad51 showed that His352 occupies an important position at the filament interface, where it could relay signals between subunits and active sites. To investigate, we characterized yeast Rad51 H352A and H352Y mutants, and solved the structure of H352Y. H352A forms catalytically competent but salt-labile complexes on ssDNA. In contrast, H352Y forms salt-resistant complexes on ssDNA, but is defective in nucleotide exchange, RPA displacement and strand exchange with full-length DNA substrates. The 2.5 A crystal structure of H352Y reveals a right-handed helical filament in a high-pitch (130 A) conformation with P6(1) symmetry. The catalytic core and dimer interface regions of H352Y closely resemble those of DNA-bound Escherichia coli RecA protein. The H352Y mutation stabilizes Phe187 from the adjacent subunit in a position that interferes with the gamma-phosphate-binding site of the Walker A motif/P-loop, potentially explaining the limited catalysis observed. Comparison of Rad51 H352Y, RecA-DNA and related structures reveals that the presence of bound DNA correlates with the isomerization of a conserved cis peptide near Walker B to the trans configuration, which appears to prime the catalytic glutamate residue for ATP hydrolysis.
Chen
Jianhong
J
Department of Biochemistry, University of Vermont College of Medicine, Burlington, VT 05403, USA.
Villanueva
Nicolas
N
Rould
Mark A
MA
Morrical
Scott W
SW
D000255:Adenosine Triphosphate / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism; D051135:Rad51 Recombinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051716:Replication Protein A / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D004277:DNA, Single-Stranded; D009711:Nucleotides; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; D000255:Adenosine Triphosphate; D004247:DNA; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase