The Human Rad51 K133A Mutant Is Functional for DNA Double-Strand Break Repair in Human Cells

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.

Green fluorescent protein fused to the C terminus of RAD51 specifically interferes with secondary DNA binding by the RAD51-ssDNA complex

“…Our biochemical results with the rice and human RAD51-GFP proteins are quite compatible with a previous genetic study involving a plant, A. thaliana (Da Ines et al, 2013), and with complementation experiments involving RAD51 -/-chicken DT40 cells or RAD51-knockdown human cells producing human RAD51-GFP (Yu et al, 2003;Forget et al, 2007). These Fig.…”

“…However, A. thaliana RAD51-GFP does not complement the mitotic DNA repair defect in the rad51/rad51 plants (Da Ines et al, 2013), consistent with earlier reports that human RAD51-GFP is also defective in mitotic DNA repair (Yu et al, 2003;Forget et al, 2007). These findings suggest that the GFP fused to the C terminus of RAD51 specifically interferes with the mitotic DNA repair function of RAD51.…”

“…Similarly, human RAD51-GFP is reportedly defective in mitotic DNA repair, although when GFP is fused to the N terminus, RAD51 functions normally (Pellegrini et al, 2002;Yu et al, 2003;Forget et al, 2007). However, the mechanism by which the C-terminal GFP of RAD51 interferes with mitotic DNA repair, without affecting the meiotic function of RAD51, has not been clarified.…”

RAD51 Mutants Cause Replication Defects and Chromosomal Instability

“…Cells are sensitive to changes to the dose of wild-type (WT) and mutant RAD51, limiting most RAD51 studies to in vitro assays or short-time-course, tissue culture assays without the opportunity to observe the long-term effects of defective RAD51 expressed at physiological levels (29). As expected, ectopic expression of RAD51 K133A and RAD51 K133R proved extremely toxic to cells, making it difficult to generate euploid cells that stably express these mutant proteins at physiological levels, especially the K133A mutant (19,31,43,54,64). For the K133A mutant, only hybridoma cells that stably express a FLAG-tagged hRAD51 K133A have been generated (33,54).…”

“…Thus, the K133A mutant is biochemically more severe than the K133R mutant. These mutants do not affect RAD51-protein interactions and have little effect on the equilibrium binding affinity of RAD51 to ssDNA (19). However, ATP binding is critical for assembly and stabilization of a catalytically active nucleoprotein filament, while ATP hydrolysis promotes filament disassembly and RAD51 release from DNA (12).…”