Measurement of meiotic and mitotic S-phase lengths. (A) Progression of DNA replication was monitored by FACS analysis of samples collected at specified time points during meiotic (SPM) and mitotic (YPD) time courses. Cells progress from an unreplicated state indicated by their presence in a single peak of fluorescence (2C), to a state in which bulk DNA replication is complete, as indicated by movement into a second peak of twice the fluorescence intensity of the first peak (4C). (B) The fraction of cells undergoing DNA replication at each time point was evaluated from the corresponding FACS profile. The fractional area under the indicated rectangle was taken to represent the fraction of cells in S phase; the fractional area under the indicated triangle, doubled, was taken to represent the fraction of cells in G₂/M. The appropriateness of these approximations is discussed in Materials and Methods. (C) Fraction of cells undergoing DNA replication at each time point in A (SPM and YPD + Noc cultures) is estimated as in Materials and Methods and plotted as a function of time. The lengths of mitotic (17.0 min) and meiotic (60.9 min) S phases were calculated as described in the text (also see Materials and Methods). (D) Cumulative curves for mitotic and meiotic DNA replication. The fraction of cells that have entered (solid line) and exited (broken line) S phase at each time point is calculated (Materials and Methods) as a function of time. The times at which 50% of active cells have entered mitotic (75 min) and meiotic (165 min) S phases are indicated. The lengths of mitotic (17 min) and meiotic (60.9 min) S phases is the time distance between the Entering S and Exiting S lines as depicted.

Experimental system. (A) NKY3000 is a self-diploidized spore clone of NKY730. A panel of homothallic heterozygous strains were generated by standard yeast transformation procedures. Each heterozygotic derivative of NKY3000 was sporulated and the four haploid spore clones from the same ascus were allowed to undergo self-diploidization. The resulting four diploid strains, two wild type and two mutant, were analyzed simultaneously during parallel synchronous meiotic time courses. (B) Distribution of wild-type meiotic S-phase lengths. (Left) The distribution of S-phase lengths observed for pairs of wild-type spore clones derived as described in A. The mean and s.d., 76.7 ± 18.9 min, is of 34 measurements from 17 different experiments. (Right) The variation between the two sibling cultures examined in parallel in a single experiment. Average length of S phase for each pair of wild-type cultures from a single experiment is summarized below.

Meiotic S-phase is shorter in spo11Δ. (A) Progression of meiotic DNA replication in two wild-type (○,□) and two spo11Δ (●,█) strains derived from NKY3002. (B) Lengths of S phases in wild type and spo11Δ. (see Fig. 1 and text for details). (C) The fraction of cells that have entered (solid line) and exited (broken line) S phase is calculated for the four cultures in A (Materials and Methods). Arrows indicate the time at which 50% of the active cells have entered S phase in each culture. (D) Lengths of meiotic S phase in wild-type and spo11Δ cultures from three different experiments. Open and solid symbols represent the calculated lengths of meiotic S phase in wild type and spo11Δ, respectively. Results from A are summarized in experiment 1. NKY3002 was analyzed for experiments 1 and 2, and NKY3196 for experiment 3. For experiment 2, one of the two wild-type and spo11Δ spore clones was analyzed in duplicate giving rise to a total of six cultures analyzed. Also shown are the average lengths of meiotic S phase for wild-type and mutant spore clones in each experiment. (E) The relative lengths of meiotic S phase in spo11Δ cultures (see text for detail). (Left) Each shaded oval corresponds to a specific ● and █ in D. (Right) Σ1–3 is the distribution of normalized S-phase length in seven spo11Δ cultures. The mean and standard deviation are used in a one-sample t-test; the confidence level that the mean value for spo11Δ (0.771) is different from that of the wild type (1.0) is >99.5%. (F) Kinetics of entry into meiotic S phase is comparable in SPO11 and spo11Δ strains. The time at which 50% of active cells have entered S-phase in each SPO11 and spo11Δ culture of the above-mentioned three independent experiments are summarized. Mean values of seven SPO11 and spo11Δ cultures are shown (right) (Σ1–3).

Relative lengths of meiotic S-phase in various mutants. (A–D) Lengths of meiotic S-phase in various mutants relative to wild type (defined as 1.0) are calculated as described in Fig. 3E. The number of circles in each strain represents the number of cultures analyzed. (A) (spo11Δ) NKY3002 and NKY3196; (spo11–Y135F) NKY3001. Analysis of NKY3197 indicated that HA-epitope tagging of Spo11p did not affect the timely occurrence of major events during meiosis, including meiotic S phase (data not shown). (B) (rec102Δ) NKY3135; (red1Δ) NKY3190; (hop1Δ) NKY3191. (C) (rec8Δ) NKY3157; (rec8Δ spo11Δ) NKY3194.

DSB-independent homolog pairing in SPO11, spo11Δ, and spo11–Y135F. Synchronous meiotic cultures for NKY1992 (SPO11; A), BWY168A (spo11Δ; B), and BWY119 (spo11–Y135F; C) were set up as described in Materials and Methods. Samples were harvested at specified time points and the extent of homolog pairing is measured by FISH analysis (Weiner and Kleckner 1994). Two homologs are defined as paired if the distance between them, visualized by a fluorescence labeled probe, is ≤0.7 μm (Weiner and Kleckner 1994). The kinetics of loss and re-establishment of pairing, (○) as well as the entry into (solid line) and exit from meiotic (broken line) S-phase are summarized.