Single-molecule analysis reveals cooperative stimulation of Rad51 filament nucleation and growth by mediator proteins

Abstract

Summary Homologous recombination (HR) is an essential DNA double-strand break (DSB) repair mechanism, which is frequently inactivated in cancer. During HR, RAD51 forms nucleoprotein filaments on RPA-coated, resected DNA and catalyzes strand invasion into homologous duplex DNA. How RAD51 displaces RPA and assembles into long HR-proficient filaments remains uncertain. Here, we employed single-molecule imaging to investigate the mechanism of nematode RAD-51 filament growth in the presence of BRC-2 (BRCA2) and RAD-51 paralogs, RFS-1/RIP-1. BRC-2 nucleates RAD-51 on RPA-coated DNA, whereas RFS-1/RIP-1 acts as a “chaperone” to promote 3′ to 5′ filament growth via highly dynamic engagement with 5′ filament ends. Inhibiting ATPase or mutation in the RFS-1 Walker box leads to RFS-1/RIP-1 retention on RAD-51 filaments and hinders growth. The rfs-1 Walker box mutants display sensitivity to DNA damage and accumulate RAD-51 complexes non-functional for HR in vivo . Our work reveals the mechanism of RAD-51 nucleation and filament growth in the presence of recombination mediators.

Generation of versatile ss-dsDNA hybrid substrates for single-molecule analysis

“…Its bacterial homolog, RecA, has been shown to grow on ssDNA bidirectionally with 2-fold kinetic preference in a 5’ to 3’ direction along the ssDNA backbone ( Bell et al., 2012 ). To determine the growth polarity of nematode RAD-51 filaments ( Belan et al., 2021 ), λ2/5 gDNA substrate will be used as described later. It should be noted, however, that gDNA substrates described in this protocol have a wide variety of usage for different enzymatic reactions including helicase unwinding, 3’ end extension by polymerases, nucleolytic processing, etc.…”

“…RAD-51 displacement should be evident as the loss of blue fluorescence signal. In the absence of RAD-51, minimal loss of RPA-eGFP signal should be present for at least 20 min ( Belan et al., 2021 ) as RPA binds very tightly to ssDNA ( Ma et al., 2017 ). If rapid fluorescence loss is observed, this is likely due to extensive photobleaching - fresh batch of PCA/PCD should be prepared.…”

“…Finally, we demonstrate the utility of these substrates for SM analysis of bidirectional growth of RAD-51-ssDNA filaments. For complete details on the use and execution of this protocol, please refer to Belan et al. (2021) .…”

“…For complete details on the use and execution of this protocol, please refer to Belan et al. (2021) .…”

Elucidating Recombination Mediator Function Using Biophysical Tools

“…A recent single-molecule imaging study demonstrated that BRC-2 (BRCA2) and Rad51 paralogs, RFS/RIP-1, stimulate nematode Rad51 filament growth [130]. Further, Belan and co-workers showed that BRC-2 and RFS/RIP-1, synergistically, can even further increase the efficiency in Rad51 filament assembly.…”