Figure 7.
Defective DSB repair in dmc1-E157K and dmc1-D311K mutants during meiosis
(A) Cells from (Figure S9) were treated with psoralen to crosslink DNA for the physical analysis of recombination at the HIS4-LEU2 locus. JMs, joint molecules; P1, parental DNA 1; P2, parental DNA 2; CO1 and CO2, reciprocal recombinants from P1 and P2; DSBs, double-strand breaks; asterisk indicates ectopic recombination. The image shown is representative of two independent experiments.
(B) Dynamics of double strand break (DSB), joint molecule (JM), and crossover (CO) accumulation were quantified as fractions of the total lane signal from (A) and a biological replicate. Plotted values show the mean of two independent experiments; error bars represent the range.
(C) Kinetics of meiotic progression from strains in (A and Figure S9) determined by spindle morphology. Spindles were visualized by anti-tubulin immunofluorescent staining; meiosis I cells had a single bipolar spindle (MI spindle, blue), whereas meiosis II cells had two bipolar spindles (MII spindle, red). Plotted values show the mean of two independent experiments with error bars representing range; 200 cells were analyzed per time point in both experiments.
(D) Immunofluorescence analysis of anaphase I and metaphase II from WT and dmc1-D311K mutants. Cells were stained with anti-α-tubulin and DAPI. Representative images of anaphase I (Ana I) and metaphase II (Meta II) nuclear division are shown (left panel) and the frequency of incomplete nuclear division at these stages in WT and dmc1-D311K strains (right panel). Anaphase I nuclear division is considered complete when two fully separated DNA masses can be distinguished. Cells with two short bipolar spindles, in metaphase II, that contain DNA bridges between the two nuclei are inferred to have failed to fully undergo anaphase I. Mean values from two independent experiments are plotted; error bars represent the range. The number of analyzed anaphase I cells was n1 = 25 and n2 = 17 for WT and n1 = 20 and n2 = 12 for dmc1-D311K. For metaphase II analysis, 42–44 WT cells and 21 dmc1-D311K cells were analyzed in each experiment.