The human Rad51 K133A mutant is functional for DNA double-strand break repair in human cells.
The human Rad51 protein requires ATP for the catalysis of DNA strand exchange, as do all Rad51 and RecA-like recombinases. However, understanding the specific mechanistic requirements for ATP binding and hydrolysis has been complicated by the fact that ATP appears to have distinctly different effects on the functional properties of human Rad51 versus yeast Rad51 and bacterial RecA. Here we use RNAi methods to test the function of two ATP binding site mutants, K133R and K133A, in human cells. Unexpectedly, we find that the K133A mutant is functional for repair of DNA double-strand breaks when endogenous Rad51 is depleted. We also find that the K133A protein maintains wild-type-like DNA binding activity and interactions with Brca2 and Xrcc3, properties that undoubtedly promote its DNA repair capability in the cell-based assay used here. Although a Lys to Ala substitution in the Walker A motif is commonly assumed to prevent ATP binding, we show that the K133A protein binds ATP, but with an affinity approximately 100-fold lower than that of wild-type Rad51. Our data suggest that ATP binding and release without hydrolysis by the K133A protein act as a mechanistic surrogate in a catalytic process that applies to all RecA-like recombinases. ATP binding promotes assembly and stabilization of a catalytically active nucleoprotein filament, while ATP hydrolysis promotes filament disassembly and release from DNA.
Forget
Anthony L
AL
Department of Biochemistry and Molecular Pharmacology, Aaron Lazare Research Building, 364 Plantation Street, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Loftus
Matthew S
MS
McGrew
Dharia A
DA
Bennett
Brian T
BT
Knight
Kendall L
KL
D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D002460:Cell Line; D004260:DNA Repair / Q000502:physiology*; D006801:Humans; D034622:RNA Interference; D051135:Rad51 Recombinase / Q000235:genetics* / Q000502:physiology*
D000255:Adenosine Triphosphate; C495153:RAD51 protein, human; D051135:Rad51 Recombinase