Abstract
The Rad51 and Rad54 proteins play important roles during homologous recombination in eukaryotes. Rad51 forms a nucleoprotein filament on single-stranded DNA and performs the initial steps of double strand break repair. Rad54 belongs to the Swi2/Snf2 family of ATP-dependent DNA translocases. We previously showed that Rad54 promotes branch migration of Holliday junctions. Here we find that human Rad51 (hRad51) significantly stimulates the branch migration activity of hRad54. The stimulation appears to be evolutionarily conserved, as yeast Rad51 also stimulates the branch migration activity of yeast Rad54. We further investigated the mechanism of this stimulation. Our results demonstrate that the stimulation of hRad54-promoted branch migration by hRad51 is driven by specific protein-protein interactions, and the active form of the hRad51 filament is more stimulatory than the inactive one. The current results support the hypothesis that the hRad51 conformation state has a strong effect on interaction with hRad54 and ultimately on the function of hRad54 in homologous recombination.
Highlights
24698 JOURNAL OF BIOLOGICAL CHEMISTRY gous dsDNA and DNA strand exchange, which lead to formation of D-loops, a key intermediate of Homologous recombination (HR)
What is the function of Rad54 branch migration (BM) activity in homologous recombination? We previously suggested hRad54 BM activity can promote dissociation of D-loops, a step required for further re-joining of the broken DNA ends along the synthesis-dependent strand annealing (SDSA) mechanism of HR
Rad51 Protein Efficiently Stimulates 3-Strand and 4-Strand BM Activity of Cognate Rad54 Protein—Previously, we showed that hRad54 can disrupt native D-loops containing human Rad51 (hRad51) protein through its ATPase-dependent BM activity [23]
Summary
24698 JOURNAL OF BIOLOGICAL CHEMISTRY gous dsDNA and DNA strand exchange, which lead to formation of D-loops, a key intermediate of HR. Thin Layer Chromatography (TLC) ATPase Assay— 4-Strand PX-junction complexes in the presence or absence of hRad51 were prepared as described above for the BM assay in standard buffer containing CaCl2 at indicated concentrations and 20 nCi of [␥-32P]ATP.
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