Fig. 5. Ethanol affects replication rate.

a Ethanol weakly activates the replication checkpoint. Cells expressing YFP-Sml1 (IG101-12D) were exposed to 0 or 6% EtOH for 2 h and imaged using fluorescence microscopy. Ethanol-exposed cells display decreased Sml1 levels. Data represent average fluorescence intensities of individual cells. Error bars represent 95% confidence intervals. N = 471 cells for 0% ethanol and N = 198 cells for 6% ethanol. Statistical significance was assessed using a two-tailed unpaired t-test with Welch’s correction. ***P < 0.001. Specifically, p-value < 0.0001. AU, arbitrary units. b Ethanol increases cellular Rnr3 protein levels. Cells expressing YFP-Rnr3 (W6986-1B) were exposed to 0 or 6% EtOH for 2 h or 0.03% MMS for 1 h and imaged using fluorescence microscopy. N = 241, 374, and 123 for 0% ethanol, 6% ethanol, and MMS, respectively. Data represent average fluorescence intensity; error bars represent 95% confidence intervals. Statistical significance was assessed using a two-tailed unpaired t-test with Welch’s correction. ***P < 0.001. Specifically, p-value < 0.0001. AU, arbitrary units. c Cell cycle progression is slower in ethanol-exposed cells. Wild‐type cells were arrested in G₁ with α‐factor and were released synchronously into S phase with the addition of pronase, in medium containing 0 or 6% ethanol. Cells were collected at the indicated time points. DNA content was assessed using flow cytometry after PI staining. The 1C peak corresponds to cells in the G₀/G₁ phase. The 2C peak corresponds to cells in the G₂/M phase. d, e Replication fork progression is perturbed by ethanol. Fork speed, measured after pulse incorporation of EdU and DNA combing, was analyzed in asynchronous cell cultures (strain PP2226) exposed for 2 h to 0 or 6% ethanol. For each condition, at least 329 tracts coming from 3 independent replicates were analyzed. N = 329 and 637 for 0 and 6% ethanol, respectively. The scatter dot plot depicts the distribution of EdU track lengths. Medians are shown by a red line and are indicated as data labels (red). Statistical significance was assessed using a two-tailed Mann–Whitney unpaired non-parametric t-test. ***P < 0.001. Specifically, p-value < 0.0001. e Examples of the DNA fibers (green) containing EdU tracks (red). EdU tracks are highlighted in white below each fiber. Source data for this figure are provided as a Source Data file.