Figure 5.

Calcium regulation-impaired dmc1 mutants exhibit meiotic defects

(A) CD spectra of the yeast Dmc1 protein (5 μM) with ATP (0.3 mM) and in the absence (light blue line) and presence of 1-mM CaCl₂ (light green line) or 5-mM CaCl₂ (green line).

(B) Graphical representation of DNA binding of Dmc1 mutants (0.25, 0.5, and 1 μM) to 5′-fluorescently labeled ssDNA (0.9-μM nucleotides) in the presence of 1-mM ATP and 1-mM MgCl₂ analyzed by gel-based assay. The error bars represent the standard deviation from three independent experiments.

(C) Graphical representation of DNA binding of Dmc1 mutants (0.25, 0.5, and 1 μM) to 5′-fluorescently labeled ssDNA (0.9-μM nucleotides) in the presence of 1-mM ATP and 1-mM CaCl₂ analyzed by gel-based assay. The error bars represent the standard deviation from three independent experiments.

(D) Western blot analysis of Dmc1 expression from synchronized meiotic culture time points in wild-type and mutant strains. Pgk1 protein was used as a loading control. Asterisk indicates an unspecific band.

(E) DAPI analysis of meiotic divisions in dmc1 mutants. Cells were harvested after 10 h after synchronous induction of meiosis and the percentage of MI and MII cells was determined using fluorescent microscopy of fixed DAPI-stained nuclei. At least 140 cells were counted for each strain. Average values from minimum three independent cultures for each strain were plotted with error bars representing standard deviation (SD). Statistical significance was calculated using unpaired t-test, n.s. not significant.

(F) Spore viability was measured after microdissection of tetrads from diploid strains carrying one copy of DMC1 and one copy of DMC1, dmc1-D311K, dmc1-E157K, or dmc1Δ. Plotted values indicate the mean with standard deviation (two-tailed unpaired t-test; ∗p < 0.05) from three independent experiments. In sum, 216 spores were analyzed per genotype in three independent experiments. N/A, non-available as DMC1/dmc1-E157K cells failed to sporulate. See also Figures S5–S7.