Speaker
Description
Programmed double-strand breaks (DSBs) are essential in meiosis to generate genetic diversity through homologous recombination (HR). A critical step in HR involves the assembly of Dmc1 recombinases on the single-stranded DNA (ssDNA) bound by the abundant Replication Protein A (RPA) proteins. This rate-limited process is regulated by accessory proteins such as Mei5-Sae3 in budding yeast (Saccharomyces cerevisiae). Under physiological conditions, Dmc1 likely encounters ssDNA containing multiple RPA molecules with each ScRPA potentially adopting different DNA binding modes (30-nt, 20-nt, and 10-nt). How Mei5-Sae3 mediates Dmc1 assembly on ssDNA containing multiple RPA molecules remains poorly understood. Here, we employed single-molecule photobleaching and colocalization to define the RPA displacement efficiency by Dmc1 and Mei5-Sae3. While bulk experiment results have shown that Mei5-Sae3 can stimulate Dmc1 to displace multiple RPA from long ssDNA template, our results demonstrate that the 20-nt binding mode of RPA is significantly more resistant to displacement compared to the 30-nt mode. These findings suggest that the RPA in 20-nt mode functions as a structural barrier or an inhibitory state that hinders Dmc1 filament assembly. We propose a regulatory model in which high RPA concentrations increase the prevalence of the 20-nt mode, thereby modulating the Dmc1-mediated recombination.
