Abstract
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.
Highlights
Homologous recombination (HR) uses a homologous template to accurately repair DNA double-strand breaks and stalled replication forks to maintain genome stability
We first verify that Rad[51] filaments are well formed on the 5 ́ DNA junction and that the supercoiled donor DNA is well spread on the electron microscopy (EM) grids (Fig. 1b)
It was an unanticipated finding that yeast Rad51-single-stranded DNA (ssDNA) filaments do not interact autonomously with dsDNA but do so robustly in the presence of Rad[54] (Fig. 5b)
Summary
Homologous recombination (HR) uses a homologous template to accurately repair DNA double-strand breaks and stalled replication forks to maintain genome stability. Rad[51] nucleoprotein filaments probe and interact with dsDNA, forming the synaptic complex that is stabilized on a homologous sequence. We propose that Rad51/Rad[54] form a functional unit operating in homology search, synaptic complex and D-loop formation. The RecA/RadA/Rad[51] family of proteins conducts the signature reactions of homology search and DNA strand invasion during HR. These recombinase scaffolds are nucleotide cofactormodulated proteins that form right-handed helical filaments on single-stranded DNA (ssDNA)[4]. Homology search relies on probing tracts of eight-nucleotide microhomology based on the transient interactions between the stretched single-stranded DNA within filament and bases in a locally melted or stretched DNA duplex[9,10,11,12,13,14,15]
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