Fig. 1.

Hop2-Mnd1 stimulates Dmc1-mediated strand exchange, leading to robust accumulation of hyper JMs. (A) Schematic of the in vitro strand exchange assay using PhiX174 DNA as substrates. (B) css was first incubated with Dmc1, then mixed with Hop2-Mnd1, RPA, and finally with lds, followed by a further incubation for 2 h at 30 °C. The products were analyzed by agarose gel electrophoresis. The signal denoted with asterisks corresponds to “hyper JMs,” which are defined as JMs bigger than those normally formed in reactions containing Swi5-Sfr1. (C) Time course analysis of Dmc1-mediated strand exchange promoted by either Hop2-Mnd1 or Swi5-Sfr1. (D) Synergistic activation of Dmc1 by Hop2-Mnd1 and Swi5-Sfr1. Dmc1 was preincubated with css, then mixed with the indicated auxiliary factor(s) and lds, and the reaction was incubated for 1 h at 30 °C. In B–D, the following concentrations of substrates were used: css, 10 µM; Dmc1, 5 µM; lds, 10 µM; RPA, 1 µM. In C and D, Swi5-Sfr1 and Hop2-Mnd1 were used at concentrations of 0.5 µM and 0.25 µM, respectively. For the graphs in B–D, mean values ± SD from three independent experiments are shown.