Abstract
The RAD51 recombinase assembles as helical nucleoprotein filaments on single-stranded DNA (ssDNA) and mediates invasion and strand exchange with homologous duplex DNA (dsDNA) during homologous recombination (HR), as well as protection and restart of stalled replication forks. Strand invasion by RAD51-ssDNA complexes depends on ATP binding. However, RAD51 can bind ssDNA in non-productive ADP-bound or nucleotide-free states, and ATP-RAD51-ssDNA complexes hydrolyse ATP over time. Here, we define unappreciated mechanisms by which the RAD51 paralog complex RFS-1/RIP-1 limits the accumulation of RAD-51-ssDNA complexes with unfavorable nucleotide content. We find RAD51 paralogs promote the turnover of ADP-bound RAD-51 from ssDNA, in striking contrast to their ability to stabilize productive ATP-bound RAD-51 nucleoprotein filaments. In addition, RFS-1/RIP-1 inhibits binding of nucleotide-free RAD-51 to ssDNA. We propose that ‘nucleotide proofreading’ activities of RAD51 paralogs co-operate to ensure the enrichment of active, ATP-bound RAD-51 filaments on ssDNA to promote HR.
Highlights
The RAD51 recombinase assembles as helical nucleoprotein filaments on single-stranded DNA and mediates invasion and strand exchange with homologous duplex DNA during homologous recombination (HR), as well as protection and restart of stalled replication forks
RFS-1/RIP-1 stimulates RAD-51 binding to single-stranded DNA (ssDNA) in the presence of nucleotides
In our previous studies on RFS-1/RIP-1, we used stopped-flow techniques and 5′-Cy3 fluorescentlylabelled oligonucleotides to study the influence of RFS-1/RIP-1 on RAD-51-ssDNA filaments in real-time in the presence of ATP
Summary
The RAD51 recombinase assembles as helical nucleoprotein filaments on single-stranded DNA (ssDNA) and mediates invasion and strand exchange with homologous duplex DNA (dsDNA) during homologous recombination (HR), as well as protection and restart of stalled replication forks. We previously identified and characterized a RAD51 paralog complex from nematodes, RFS-1/RIP-1, discovering it has a critical role in stabilizing RAD-51-ssDNA filaments by reducing RAD-51 dissociation rate. RAD51 slowly hydrolyzes ATP in the presence of DNA10,11, meaning the presynaptic complex is likely to have a finite lifetime as an active recombinase, becoming non-functional if the ratio of ATP:ADP falls below the threshold required for strand invasion activity. It was shown that the enzymatic recombinogenic activity of RAD51 is not required to protect stalled forks[16], but expression of either ATP binding or hydrolysis-deficient RAD51 mutants causes fork stalling and increases gross chromosomal rearrangements[17] It is still not clear precisely how ATP hydrolysis or nucleotide binding affects RAD51’s role in replication
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