Defective homologue juxtaposition in hop2, mnd1, and dmc1 mutants. Typical fluorescence micrograph of meiotic nuclear spreads stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue) and GFP (white) at t = 0h(A) and t = 4 h (B) after sporulation medium transfer. (C) Quantitative analysis of homologue juxtaposition. For each strain, pairing analysis was carried out in at least three independent experiments. In each experiment, 100 nuclei were scored at the indicated time points. The average frequency of pairing is shown. (D) Timing of the MI. The percentage of cells that had executed the MI was scored as the percentage of cells containing two, three, or four DAPI-staining bodies.

Purification and size determination of Hop2 and Mnd1–His-6 protein complexes. (A) Purification of Mnd1–His-6 (M) and Hop2 (H) protein using Co(II) resin. Lanes 1 and 2 contain total cell lysates after and before isopropyl β-d-thiogalactoside induction, respectively. Lane 3 contains the soluble fraction of the total cell lysates. Lane 4 contains the fraction that failed to bind the Co(II) resin. Lane 5 represents 4 μg of purified H2M1 complexes. (B) Glutaraldehyde cross-linking of the H2M1 complex. Proteins were incubated with 0–8 mM glutaraldehyde for 10 min. The samples were separated by 10% reducing SDS/PAGE, and the protein was detected by blotting with anti-His-6 antibodies. Positions of molecular mass markers are shown in kDa.

H2M1 complexes associate with dsDNA. (A) φX174 dsDNA (15 μMin bp) and ssDNA (30 μM in nt) were incubated with H2M1 protein (0–1.5 μM). Nucleoprotein supercomplexes were separated on a 0.8% agarose gel, stained with ethidium bromide, and visualized by UV transillumination. (B) H2M1 complexes preferentially bind duplex DNA. DNA binding specificity of H2M1 complexes was determined by mixing circular φX174 dsDNA with various dinucleotide repeat oligonucleotides. The melting temperatures (Tm) and ssDNA contents of these dinucleotide repeat oligonucleotides have been indicated (40). (C) AFM images of H2M1 protein (1), 872-bp dsDNA (2), and H2M1–dsDNA complexes (3–6).

H2M1 complexes promote Dmc1-mediated homologous assimilation of ssDNA into dsDNA. (A, lanes a–h) Strand assimilation is homology dependent. PA1655 and PA1656 were ³²P-labeled with T4 polynucleotide kinase. PA1655 is homologous to pUC18-Kan; P1656 is homologous to GW1. Reaction mixtures were deproteinated by addition of 0.25% SDS and 0.25 mg/ml proteinase K. The assimilated and free oligonucleotides were separated on a 0.8% agarose gel and visualized by a PhosphoImager. (A, lanes i–o) H2M1 complexes enhance the Dmc1-mediated strand assimilation activity. pUC18-Kan was incubated with PA1655, adenosine 5′-[β,γ-imido]triphosphate, and Dmc1. (B) Quantitative results of two independent experiments as shown in A (lanes j–o). Quantitative data at zero Dmc1 protein concentration (A, lane i) were used for background correction (i.e., 0% D-loop).