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. 2018 Feb 1;7:e33309. doi: 10.7554/eLife.33309

Figure 1. Mcm2–7 loading onto replication origins is inhibited during the MI–MII transition.

(A) The DNA replication program and chromosome segregation program are uncoupled during the MI–MII transition. Relative CDK activity at various stages of the meiotic cell cycle are shown (Carlile and Amon, 2008). The dashed boxes highlight the oscillations of low-to-high CDK activity during meiosis, and show the discrepancy between CDK-regulation of SPB duplication and DNA replication. See text for details. (B) ORC is bound to origins of replication throughout the meiotic divisions. The strain yDP71 was put through meiosis. ChIP–qPCR was used to detect ORC binding at the early–firing origin ARS1 (top graph, dark blue), the late–firing origin ARS1413 (top graph, light blue), and at the re–replication prone origins ARS305 (bottom graph, dark blue) and ARS418 (bottom graph, light blue). The time after transfer into sporulation medium and the associated meiotic stages are indicated below each lane. For cell–cycle stage quantification for this experiment, see Figure 1—figure supplement 1A. The peak % of input DNA immunoprecipitated (set to arbitrary unit (A.U.) =1.0) was 9.1% for ARS1, 2.2% for ARS1413, 46.7% for ARS305, and 10.9% for ARS418. (C) Mcm2–7 is bound to origins of replication in G1 phase but does not reassociate with origins during or between the meiotic divisions. The strain yDP71 was put through meiosis. ChIP–qPCR was used to detect Mcm2–7 binding at the early–firing origin ARS1 (top graph, red), the late–firing origin ARS1413 (top graph, orange), and at the re–replication prone origins ARS305 (bottom graph, red) and ARS418 (bottom graph, orange). The time after transfer into sporulation medium and the associated meiotic stages are indicated below each lane. For cell–cycle stage quantification for this experiment, see Figure 1—figure supplement 1B. The peak % of input DNA immunoprecipitated (set to A.U. = 1.0) was 7.4% for ARS1, 5.0% for ARS1413, 35% for ARS305, and 19.5% for ARS418.

Figure 1—source data 1. Raw values used for the quantification of Figure 1B and C.
elife-33309-fig1-data1.xlsx (52.6KB, xlsx)
DOI: 10.7554/eLife.33309.006

Figure 1.

Figure 1—figure supplement 1. Cells from Figure 1 proceeded synchronously through meiosis.

Figure 1—figure supplement 1.

(A) Cell–cycle stage quantification for Figure 1B. (B) Cell–cycle stage quantification for Figures 1C and 2A. (C) Representative micrographs from a separate experiment showing the peak time point of metaphase I, anaphase I, metaphase II, and anaphase II. Insets show an enlarged cell from that cell-cycle stage. Percent of cells in that stage written above the micrographs (based on counting 100 cells). Top: tubulin, middle: DAPI, bottom: merge (red = tubulin, blue = DAPI).

Figure 1—figure supplement 2. Both ORC and Mcm2-7 associate specifically with origins of replication compared to non-origin DNA.

Figure 1—figure supplement 2.

(A) Enrichment of DNA immunoprecipitated (as a percent of input) was compared at different origins of replication relative to non-origin control DNA (URA3 locus). For ORC, the G2 ChIP was compared between each replication origin and URA3. For Mcm2-7, the G1 ChIP was compared between each replication origin and URA3.

Figure 1—figure supplement 3. Mcm2–7, Cdt1, and Orc1 proteins are present throughout meiosis.

Figure 1—figure supplement 3.

(A) Top: Immunoblots showing that Mcm2, Mcm3, Mcm6, Mcm7, Cdt1, and Orc1 are present at constant levels throughout meiosis (strain yDP71). Pgk1 and Ponceau are shown as loading controls. Bottom: Cell–cycle stage quantification.