RPA resolves conflicting activities of accessory proteins during reconstitution of Dmc1-mediated meiotic recombination

Dmc1 catalyzes homology search and strand exchange during meiotic recombination in budding yeast and many other organisms including humans. Here we reconstitute Dmc1 recombination in vitro using six purified proteins including Dmc1 and its accessory proteins RPA, Rad51, Rdh54/Tid1, Mei5-Sae3, and Hop2-Mnd1 to promote D-loop formation between ssDNA and dsDNA substrates. Each accessory protein contributed to Dmc1s activity, with the combination of all six proteins yielding optimal activity. The ssDNA binding protein RPA plays multiple roles in stimulating Dmc1s activity including by overcoming inhibitory effects of ssDNA secondary structure on D-loop reactions, and by stabilizing and elongating D-loops. In addition, we demonstrate that RPA limits inhibitory interactions of Hop2-Mnd1 and Rdh54/Tid1 that otherwise occur during assembly of Dmc1-ssDNA nucleoprotein filaments. Finally, we report interactions between the proteins employed in the biochemical reconstitution including a direct interaction between Rad51 and Dmc1 that is enhanced by Mei5-Sae3.