Functional interactions of meiotic recombination factors Rdh54 and Dmc1

Abstract

Genetic studies in budding and fission yeasts have provided evidence that Rdh54, a Swi2/Snf2-like factor, synergizes with the Dmc1 recombinase to mediate inter-homologue recombination during meiosis. Rdh54 associates with Dmc1 in the yeast two-hybrid assay, but whether the Rdh54-Dmc1 interaction is direct and the manner in which these two recombination factors may functionally co-operate to accomplish their biological task have not yet been defined. Here, using purified Schizosaccharomyces pombe proteins, we demonstrate complex formation between Rdh54 and Dmc1 and enhancement of the recombinase activity of Dmc1 by Rdh54. Consistent with published cytological and chromatin immunoprecipitation data that implicate Rdh54 in preventing the non-specific association of Dmc1 with chromatin, we show here that Rdh54 mediates the efficient removal of Dmc1 from dsDNA. These functional attributes of Rdh54 are reliant on its ATPase function. The results presented herein provide valuable information concerning the Rdh54-Dmc1 protein pair that is germane for understanding their role in meiotic recombination. The biochemical systems established in this study should be useful for the continuing dissection of the action mechanism of Rdh54 and Dmc1.

Figure 1. SpDmc1 binding and promotion of the D-loop reaction by purified SpRdh54

(A) Purified SpRdh54 and SpDmc1 (1 μg) were analyzed by SDS-PAGE with Coomassie Blue staining. (B) SpRdh54 was mixed with Affi-Gel 15 beads or beads containing covalently conjugated BSA or SpDmc1. After washing, the bound SpRdh54 was eluted with SDS (E) and analyzed along with the supernatant (S) and wash (W) by SDS-PAGE and Coomassie Blue staining. (C) The reaction schematic of the D-loop reaction is shown in panel I. In panel II, the ability of SpDmc1 to form D-loop was tested in the absence or presence of SpRdh54 (150 and 250 nM in lanes 4 and 5, and 380 nM in lanes 2, 6, 7, and 9) or ATP, as indicated. The results were plotted in panel III.

Figure 2. Dissociation of the SpDmc1-dsDNA complex by SpRdh54

(A) The reaction schematic is shown. (B) Nucleoprotein complexes of SpDmc1 and magnetic bead-bound DNA were assembled with either ATP or AMP-PNP as nucleotide cofactor and then incubated without or with SpRdh54 (200 nM in lane 3, 400 nM in lanes 4 and 8, 600 nM in lane 5, and 750 nM in lanes 6 and 9). The supernatant and bead fractions were analyzed by SDS-PAGE and Coomassie Blue staining for their protein content. CK, creatine kinase used in the ATP regenerating system. The results were plotted.

Figure 3. Dependence of functional interactions on the Rdh54 ATPase activity

(A) ScRdh54 or its K352R variant was mixed with Affi-Gel 15 beads containing covalently conjugated BSA or SpDmc1. After washing, the bound Rdh54 or rdh54 K352 protein was eluted with SDS (E) and analyzed along with the supernatant (S) and wash (W) by SDS-PAGE and Coomassie Blue staining. (B) The ability of ScRdh54 (120, 240, and 360 nM in lanes 3-5, respectively) or its K352R variant (120, 240, and 360 nM in lanes 7 to 9, respectively) to dissociate the SpDmc1-dsDNA nucleoprotein complex, as in Figure 2. The results were plotted.