Dual regulation of Dmc1-driven DNA strand exchange by Swi5-Sfr1 activation and Rad22 inhibition

Abstract

Both ubiquitously expressed Rad51 and meiosis-specific Dmc1 are required for crossover production during meiotic recombination. The budding yeast Rad52 and its fission yeast ortholog, Rad22, are "mediators;" i.e., they help load Rad51 onto ssDNA coated with replication protein A (RPA). Here we show that the Swi5-Sfr1 complex from fission yeast is both a mediator that loads Dmc1 onto ssDNA and a direct "activator" of DNA strand exchange by Dmc1. In stark contrast, Rad22 inhibits Dmc1 action by competing for its binding to RPA-coated ssDNA. Thus, Rad22 plays dual roles in regulating meiotic recombination: activating Rad51 and inhibiting Dmc1.

Keywords: Dmc1; Rad52 mediator; crossover; homologous recombination; meiosis; negative regulation.

Figure 1.

Swi5–Sfr1 stimulates the Dmc1-driven DNA three-strand exchange reaction. (A) Schematic of the three-strand exchange reaction. (B) Strand exchange in the Dmc1-start or RPA-start reactions with various concentrations of Swi5–Sfr1. The right graph shows quantification of results from the gels on the left. (C) Time-course experiment of Dmc1-driven strand exchange reaction. Large-volume reactions (50 μL) were carried out under standard conditions. Aliquots (6.5 μL) were taken at the indicated time points. Different concentrations of Swi5–Sfr1 were added to the RPA-start reaction (open symbols); closed triangles show the results of the Dmc1-start reaction in the presence of 1 μM Swi5–Sfr1. cssDNA (10 μM), ldsDNA (10 μM), Dmc1(5 μM), and RPA (1.5 μM) were included in each experiment.

Figure 2.

Swi5–Sfr1 facilitates Dmc1 loading onto ssDNA. (A) Schematic of ssDNA pull-down assay. Purified protein mixtures (5 μM Dmc1, 1 μM RPA, and 3.5 μM Swi5–Sfr1) were incubated with 10 μM ssDNA beads in the absence or presence of 1 mM various adenine nucleotise di- or triphosphates for 15 min at 30°C. The bead-bound fractions were pulled down using a magnetic stand and analyzed by SDS-PAGE. Proteins stained with Coomassie brilliant blue R-250 were quantified with an image analyzer. (B) Dmc1 only. (C) Dmc1 and Swi5–Sfr1. (D) Dmc1 and RPA. (E) Dmc1, Swi5–Sfr1, and RPA. When two or three proteins were incubated, a premix was prepared to add them simultaneously. (F) Schematic of ssDNA bead pull-down assay for Dmc1 loading onto RPA-coated ssDNA. RPA-coated ssDNA beads were prepared by washing the incubation mixture, which included 1 μM final concentration RPA and 10 μM ssDNA beads (in terms of total nucleotides) for 20 min at 30°C. Dmc1 (5 μM) and various concentrations of Swi5–Sfr1 were added to the RPA-coated ssDNA beads, and the bead-bound and supernatant (unbound) fractions were analyzed by SDS-PAGE. (G) An image of an SDS-PAGE gel of the pull-down assay (left) and graphic presentations of bound Dmc1 (middle) and displaced RPA (right) with values from three independent experiments (mean ± SD).

Figure 3.

Swi5–Sfr1 stabilizes Dmc1 filaments. (A) Dmc1 filaments were prepared on ssDNA beads in the presence or absence of Swi5–Sfr1, and then RPA from S. pombe (Sp) or humans was added to the reaction. Bound and unbound proteins were analyzed by SDS-PAGE as in Figure 2. (B) SDS-PAGE image of the fractions (top) and graphic presentations of bound Dmc1 (middle) and displaced RPA (bottom) from three independent experiments (mean ± SD). cssDNA (10 μM), ldsDNA (10 μM), Dmc1 (5 μM), and RPA (1.5 μM) were added to each reaction.

Figure 4.

Effects of wild-type Rad22 (SpRad52) on Dmc1-driven strand exchange reaction. (A) Wild-type Rad22 protein (circles in the right panels), but not a Rad22 mutant defective in RPA binding (Rad22^{D240A–E241A}; triangles in the right panels), inhibits the RPA-start strand exchange reaction. Scheme is as in Figure 1, but Rad22 was added before Dmc1 and Swi5–Sfr1 (at time point B in B). (B) An order of addition experiment revealed that the reaction was inhibited by Rad22 before the addition of Dmc1 only under the RPA-start condition. (C) Wild-type, but not mutant, Rad22 protein inhibits Dmc1 loading onto RPA-coated ssDNA. Scheme is as in Figure 2A, but Rad22 was added simultaneously with Dmc1 and Swi5–Sfr1. (D) Rad22^D250–E251A but not Rad22^{D240A–E241A} binds to RPA, as judged by the GST pull-down assay. (I) Input; (FT) flow-through; (W) wash; (E) eluate fractions. Rad22 (1 μM) was added to the reaction 5 min after the addition of each reaction component at the indicated time points in A–D.