Figure 3. Top1-mediated Ribonucleotide Removal Affects Multiple Phenotypes.
(A) Flow cytometry analysis. In each histogram, the horizontal axis represents the fluorescence parameter and the vertical axis represents the number of cells. Plotted black lines represent the raw data and the smoothed data generated by ModFit software analysis. The gray shaded areas represent cells in G1 or G2/M phases, the striped area represent cells in S phase. (B) The percentage of cells in each stage of the cell cycle was calculated based on flow cytometry analysis of DNA content. The experiment was performed in triplicate, data are displayed as the mean % cells ± standard error. *, P = 0.023, **, P = 0.0136 (two-tailed Student’s t tests). (C) Measurement of dNTP pools. Each strain genotype was independently analyzed twice. The data displayed represents the mean total dNTP abundance ± standard error. *, P = 0.0032 (two-tailed Student’s t test). All strains harbor the pol2-M644G mutant allele. (D) A quantitative HU-survival assay was performed by plating G1-arrested cells onto YPDA agar ± 150 mM HU. The graph represents data for the indicated pol2-M644G mutant strains from three independent experiments with % survival calculated as the percentage of surviving cells compared to the untreated control (± standard error). *, P < 0.0001 (two-tailed Student’s t test). (E) Ten-fold serial dilutions of exponentially growing cells spotted onto YPDA agar plates (untreated) or exposed to 150 mM HU. (F) The specific mutation rate of 2-5 bp deletions in repeat sequences was determined by sequencing a collection of 5-FOA-resistant colonies from each strain. The orientation-dependent difference in mutation rate results from a 2-base deletion hotspot that is only observed in URA3-OR2.