PMC

(a) Immunostaining analysis of Rad51 and Dmc1 foci on meiotic chromosome spreads. Green, Rad51; Red, Dmc1. Scale Bar, 2 μm. (b) Kinetic analysis of Rad51 and Dmc1 foci. Percents of focus-positive cells (bottom) and an average number of the focus per focus-positive nucleus (top) are shown at each time point. A focus-positive nucleus is defined as spreads with more than 5 foci. Representative results are shown (n = 3).

Chromatin-immunopreciptation (ChIP) analyses of control (a, black), Psy3-3FLAG (b, blue), Rad51 (c, green), Shu1-3FLAG (d, purple), and Dmc1 (e, orange) were carried out in different strains. Binding efficiencies (% of immunoprecipitated to whole-cell lysates) of each protein to a region in the HIS4-LEU2 recombination hotspot (closed bars) and a control region of internal region of the SMC1 ORF (open bars) are shown. In some experiments, s.d. (n = 3) is shown.

(a) A top Rad51 dimer in Rad51 filament (1SZP) with 6 Rad51 protomers (green; left) was replaced with Psy3-Csm2 (blue-pink; middle) after the structural fit of the Psy3-Csm2 to the Rad51 dimer (right). (b) The position of a mass with Shu1-Shu2 dimer (grey oval) in Psy3-Csm2-Shu1-Shu2 (She et al, 2012) is placed on the putative Rad51 filament with Psy3-Csm2. Compared with the model in a, the structure is rotated by 180°. (c) An interface between yeast Rad51 and yeast Rad51 (green; left), human Rad51 and a BRC repeat of human Brca2 (right) is shown in topographic representation. A putative interface between Rad51 (green) and Csm2 (pink) is presented in the middle. Rad51-Rad51 and human Rad51-BRC is from 1SPZ and 1N0W, respectively.

(a) Schematic representation of the HIS4-LEU2 recombination hotspot assay locus for 2D assay. (b) Southern blot images of one-dimensional (1D) gel analysis of meiotic recombination in wild-type and csm2 strains. Genomic DNA samples at each time point were digested with XhoI, separated on an agarose gel, and visualized by Southern blotting. (c) Southern images of two-dimensional (2D) gel analysis of JM intermediates, single-end invasions (SEIs) and double-Holliday junctions (dHJs). No JMs are detected in csm2 dmc1 and csm2 hop2 strains (right-hand side panels), showing that strand-exchange in the csm2 mutant is completely dependent on Dmc1 and its accessory complex, Hop2(-Mnd1). (d) Temporal analysis of DSB, SEI, IH-dHJ, IS-dHJ, multi-chromatid JMs (m-JM), and crossovers are shown for wild-type (orange) and csm2 (magenta) strains Representative results are shown (n = 3). (e) A ratio of IH-dHJ to IS-dHJ in wild-type and csm2 is shown.

(a) Representative meiotic nuclei immunostained for Rad51 (green) and Dmc1 (red) in the srs2 and srs2 psy3 mutant backgrounds. (b) Average number of Rad51 (green) or Dmc1 (red) foci per focus-positive nucleus in wild-type (4 h), psy3 (5 h), srs2 (5 h) and srs2 psy3 (5 h) strains. A focus-positive nucleus is defined as having >5 foci. Representative results are shown (n =3). Statistical differences are shown in main text. (c) Timing of meiotic division I in wild-type and various mutant cells was measured by DAPI staining. (d) Southern blot images of 1D gels analysing crossovers and non-crossovers (left). Quantification of meiotic divisions, and crossover (R1) and non-crossover (R3) levels determined by Southern blot analysis. Representative results are shown (n = 3). Wild-type (open orange circles), psy3 (open blue circles), srs2 (closed orange circles) and srs2 psy3 (closed blue circles).

(a) Each 1 μg of purified fractions of Psy3-Csm2 and Shu1-Shu2 are analysed on SDS–PAGE gel and visualized with the Coomassie staining. (b) A profile of gel filtration using Superdex200 of Psy3-Csm2 (magenta) and Shu1-Shu2 (brown) are shown with size markers. (c) The binding of either Psy3-Csm2 or Shu1-Shu2 to ss- or dsDNAs (ϕX174 DNA, 135 μM) was studied by EMS assay. Protein–DNA complexes were resolved in a 0.8% agarose gel and were visualized with EtBr-staining. (d) A crystal structure of Psy3-Csm2 dimer is shown in a ribbon-presentation. Csm2, pink; Psy3, blue. (e) A stereo view of an interface between Psy3 (blue) and Csm2 (pink) is shown. Maps (2Fo-Fc) are shown at 1.5σ contour levels. (f) Structural comparison among Rad51 protomer (left), Psy3 (middle) and Csm2 (right). Homologous α-helixes and β-sheets are shown in colours. (g) Schematic comparison of domains among Rad51, Psy3 and Csm2. Two loops, L1 and L2, are DNA binding regions of Rad51 and homologous loops to L1 and L2 are shown for Psy3 and Csm2. (h) Comparison of Psy3-Csm2 with Rad51 dimer. Csm2, pink; Psy3, blue. Rad51 dimer (1SZP) is shown in green (right monomer, chain B in 1SZP) and light green (left monomer, chain C in 1SZP). Super-imposition of Rad51 with Psy3-Csm2 dimer is indicated as a stereo view. Psy3-Csm2 is simply superimposed with Rad51 dimer using COOT. (i) The interface between Psy3 and Csm2 (top) is compared with that between Rad51 (green) and Rad51 (light green) protomers (bottom).

(a) Affinity purification of Shu1-3FLAG. Fractions from mitotic and meiotic cells were analysed by silver staining. (b) Complex formation of the PCSS was examined by gel filtration of purified Shu1-3FLAG fractions. (c) Spore viability of various pcss mutants (n = 100). (d) Heteroallelic recombination between arg4-bgl and arg4-nsp alleles was quantified by the ‘return-to-growth’ assay. The graphs show the frequencies of Arg ⁺ prototrophs at 0 (left) and 24 (right) hours after the induction of meiosis (n =2). (e) Schematic representation of the HIS4-LEU2 recombination hotspot assay locus located on S. cerevisiae chromosome 3. Diagnostic XhoI (X) restriction-site polymorphisms and the position of the probe are shown. The size and composition of detected DNA species are shown below. Each line represents a DNA duplex. (f) Physical analysis of repair of meiotic double-strand breaks (lower panels) and the formation of crossovers and non-crossovers (upper panels) in meiotic recombination. (g) Quantification of crossovers (R1) and non-crossovers (R3) in f. An average of three independent time courses is shown with error-bars (s.d).

(a) Co-complex formation of Rad51 and Psy3-Csm2 on DNA was analysed in EMS assay. Different concentrations of Rad51 and Psy3-Csm2 are incubated with a FITC-labelled 60 mer ssDNA (6 μM in nucleotide) in the presence of 1 mM ATP (top), 1 mM ADP (middle) and in the absence of any nucleotides (bottom). The complexes were resolved in a 0.8% agarose containing 1 mM MgCl₂. Protein–DNA complexes were analysed with an image analyser. PC, Psy3-Csm2; R51; Rad51. (b) Antibodies against Rad51 and Psy3 were added to check the presence of each protein in the complex. The antibodies were added after the complex formation. (c) A putative interface of Rad51 (green) and Csm2 (pink) is shown with possible amino acids of Csm2 responsible to Rad51-binding. (d). Purification of Psy3-Csm2-I83A/L87A and Psy3-Csm2-I40A/I83A. One microgram of purified complexes was analysed on SDS–PAGE. (e) EMS analysis of DNA binding of Psy3-Csm2-I83A/L87A and Psy3-Csm2-I40A/I83A. (f) Effects of Psy3-Csm2-I83A/L87A and Psy3-Csm2-I40A/I83A proteins on Rad51-filament stabilization. The analysis was carried out as shown (a). (g) Different concentrations of yeast and human Rad51 were incubated with the oligo-DNA and ATP in the absence or the presence of Psy3-Csm2. Products were analysed on an agarose gel. Yeast Rad51, green: human Rad51, deep green.