In vitro role of Rad54 in Rad51-ssDNA filament-dependent homology search and synaptic complexes formation
Résumé
Homologous recombination (HR) uses a homologous template to accurately repair DNA
double-strand breaks and stalled replication forks to maintain genome stability. During
homology search, Rad51 nucleoprotein filaments probe and interact with dsDNA, forming the
synaptic complex that is stabilized on a homologous sequence. Strand intertwining leads to
the formation of a displacement-loop (D-loop). In yeast, Rad54 is essential for HR in vivo and
required for D-loop formation in vitro, but its exact role remains to be fully elucidated. Using
electron microscopy to visualize the DNA-protein complexes, here we find that Rad54 is
crucial for Rad51-mediated synaptic complex formation and homology search. The Rad54
−K341R ATPase-deficient mutant protein promotes formation of synaptic complexes but not
D-loops and leads to the accumulation of stable heterologous associations, suggesting that
the Rad54 ATPase is involved in preventing non-productive intermediates. We propose that
Rad51/Rad54 form a functional unit operating in homology search, synaptic complex and
D-loop formation.
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