Inter-subunit interactions that coordinate Rad51's activities.
Rad51 is the central catalyst of homologous recombination in eukaryotes and is thus critical for maintaining genomic integrity. Recent crystal structures of filaments formed by Rad51 and the closely related archeal RadA and eubacterial RecA proteins place the ATPase site at the protomeric interface. To test the relevance of this feature, we mutated conserved residues at this interface and examined their effects on key activities of Rad51: ssDNA-stimulated ATP hydrolysis, DNA binding, polymerization on DNA substrates and catalysis of strand-exchange reactions. Our results show that the interface seen in the crystal structures is very important for nucleoprotein filament formation. H352 and R357 of yeast Rad51 are essential for assembling the catalytically competent form of the enzyme on DNA substrates and coordinating its activities. However, contrary to some previous suggestions, neither of these residues is critical for ATP hydrolysis.
Grigorescu
Arabela A
AA
Department of Biochemistry, Molecular Biology and Cell Biology, The University of Chicago, Chicago, IL 60637, USA.
Vissers
Joseph H A
JH
Ristic
Dejan
D
Pigli
Ying Z
YZ
Lynch
Thomas W
TW
Wyman
Claire
C
Rice
Phoebe A
PA
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D004277:DNA, Single-Stranded / Q000378:metabolism; D018625:Microscopy, Atomic Force; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051135:Rad51 Recombinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D004277:DNA, Single-Stranded; D009711:Nucleotides; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase; D000251:Adenosine Triphosphatases