Figure 3. The ability of mec1‐100 cells to resume DNA replication under replicative stress is impaired by either Sgs1‐G1298R or the lack of Rad9.

• A
  Cells were arrested in G1 with α‐factor (αf) and then released in YEPD containing 0.2 M HU at time zero. After 2 h (HU), cells were transferred to medium lacking HU but containing nocodazole to prevent passage through mitosis. Aliquots of each culture were harvested at the indicated times after HU removal to determine DNA content by flow cytometry.
• B
  Cell viability. Cells were arrested in G1 with α‐factor and then released in YEPD containing 0.2 M HU at time zero. Cells taken at the indicated time points after release in HU were tested for colony‐forming units on YEPD plates. Plotted values are the mean values with error bars denoting s.d. (n = 3).
• C, D
  Immunodetection of BrdU‐pulsed DNA. Cells were arrested in G1 with α‐factor (αf) and released into YEPD containing 0.2 M HU + 25 μM BrdU. After 1 h (HU), cells were chased with 2 mM thymidine into fresh medium and samples were taken at the indicated times after chase (−HU). DNA content during the time course was measured by flow cytometry (C). BrdU‐labeled DNA was detected with anti‐BrdU antibodies (D). High molecular weight DNA molecules are indicated by an arrow.
• E
  ChIP analysis. Cells were arrested in G1 with α‐factor and then released in YEPD containing 0.2 M HU at time zero. Relative fold enrichment of Myc‐tagged DNA Polε at ARS607 and ARS305 replication origins was determined after ChIP with anti‐Myc antibodies and subsequent qPCR analysis. Plotted values are the mean values with error bars denoting s.d. (n = 3). *P < 0.05 (Student's t‐test).
• F
  Cells were arrested in G1 with α‐factor (αf) and then released in YEPD containing 0.2 M HU at time zero, followed by Western blot analysis of protein extracts with anti‐Rad53 antibodies.

Source data are available online for this figure.