Abstract
In eukaryotes, Dmc1 and Rad51 are key proteins of homologous recombination. The Swi5-Sfr1 complex in fission yeast, a conserved auxiliary factor, stimulates DNA strand exchange driven by both Dmc1 and Rad51. Interestingly, biochemical analysis suggested that Swi5-Sfr1 regulates strand exchange activities of these recombinases differently, but the mechanisms were unclear. We previously developed a real-time system to analyze Rad51-driven DNA strand exchange and identified two topologically distinct three-stranded intermediates (complex 1 (C1) and complex 2 (C2)). Swi5-Sfr1 facilitates the C1-C2 transition and releases single-stranded DNA (ssDNA) from C2, acting as a strand exchange activator. In this study, we investigated fission yeast Dmc1-driven DNA strand exchange and the role of Swi5-Sfr1 in Dmc1 activity in real-time. Kinetic analysis revealed a three-step model for the Dmc1-driven reaction, similar to that of Rad51. Although Swi5-Sfr1 stimulated the Dmc1-driven reaction, it had a weaker impact than Rad51. Furthermore, Swi5-Sfr1 enhanced the association of Dmc1 with ssDNA by promoting filament nucleus formation, acting as a mediator, unlike its role with Rad51. This stimulation mechanism also differs from that of Ca2+ or ATP analog, AMP-PNP. Our findings suggest that Swi5-Sfr1 stimulates strand exchange activities of Dmc1 and Rad51 via different reaction steps.