Figure 6. Sld2 is degraded during the meiotic divisions in a manner that depends on Cdc5- and CDK-phosphorylation sites.

(A) Sld2 protein is degraded upon entry into the meiotic divisions. Immunoblots of Sld2–13myc during meiosis from strain yDP336. The time after transfer into sporulation medium and the associated meiotic stages are indicated above each lane. For cell–cycle synchrony, refer to Figure 6—figure supplement 2A. (B–D) Mutation of either Cdc5– or CDK–phosphorylation sites on Sld2 results in stabilization of Sld2 throughout the meiotic divisions: Immunoblots of Sld2–13myc during meiosis with the following mutations: (B) Cdc5–phosphorylation sites (strain yDP473: 2TA – T122A/T143A), (C) CDK–phosphorylation sites (strain yDP642: 2SA – S128A/S138A), or (D) Cdc5– and CDK–phosphorylation sites (strain yDP644: 4A – T123A/S128A/S138A/T143A). The time after transfer into sporulation medium and the associated meiotic stages are indicated above each lane. For cell–cycle synchrony, refer to Figure 6—figure supplement 2B–2D. (E) Top: Samples from (A–D) with the peak number of cells in G2, Metaphase I, Anaphase I, Metaphase II, and Anaphase II were run side–by–side. Middle: Mean of Sld2 levels normalized to PGK1 levels from three independent experiments. Bottom: Graph of Sld2/PGK1 quantification from three independent experiments. The mean is represented by the height of the bar. Error bars represent the standard deviation.

Figure 6—figure supplement 1. Most helicase–activation proteins are present throughout the meiotic divisions.

(A–D) Top: Immunoblots for Cdc45–13myc, Sld3–13myc, Dpb11–3V5, and Psf2–3V5, respectively. The time after transfer into sporulation medium and the associated meiotic stages are indicated above each lane. Bottom: Cell–cycle stage quantification.

Figure 6—figure supplement 2. Cells from Figure 6 proceeded synchronously through meiosis.

(A–D) Cell–cycle stage quantification for Figure 6A–D, respectively.

Figure 6—figure supplement 3. Sld2-13myc and Pgk1 levels are quantifiable across a 32-fold dilution range.

(A) Sld2 and Pgk1 western blots have increased intensity with increased input. (B) Quantification of Sld2 and Pgk1 levels is similar to the expected value based on the relative amount of input.