GSBS Student Publications
Title
The human Rad51 K133A mutant is functional for DNA double-strand break repair in human cells
GSBS Program
Biochemistry & Molecular Pharmacology
Publication Date
2007-02-17
UMMS Affiliation
Graduate School of Biomedical Sciences; Department of Biochemistry and Molecular Pharmacology
Document Type
Article
Disciplines
Life Sciences | Medicine and Health Sciences
Abstract
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.
DOI of Published Version
10.1021/bi062128k
Source
Biochemistry. 2007 Mar 20;46(11):3566-75. Epub 2007 Feb 16. Link to article on publisher's site
Journal/Book/Conference Title
Biochemistry
Related Resources
PubMed ID
17302439
Repository Citation
Forget, Anthony L.; Loftus, Matthew S.; McGrew, Dharia A.; Bennett, Brian Thomas; and Knight, Kendall L., "The human Rad51 K133A mutant is functional for DNA double-strand break repair in human cells" (2007). GSBS Student Publications. 298.
https://escholarship.umassmed.edu/gsbs_sp/298