71) (Table 1; Additional file 1: Table S2). Figure 2 The rad59 mutant alleles have distinct effects on cell cycle distribution in rad27::LEU2 mutant cells. Wild-type, single and double mutant strains were grown to mid-log phase at 30°, fixed, stained with propidium iodide, and submitted to flow cytometric analysis as described in the Methods. (A) Cell cycle profiles for wild-type and rad59 mutant strains. (B) Cell cycle profiles for rad27 single and rad27 rad59 double mutants. learn more The distribution of cells with 1n and 2n DNA
content in representative cultures of each strain are depicted. (C) Cell cycle distribution for wild-type and mutant strains. Median ratios of G1 to S + G2/M cells from a minimum of five independent cultures are indicated for each strain, and 95% confidence intervals are plotted. Table 1 Doubling times in wild-type and mutant haploid cells Genotype Doubling time (min) 95% confidence interval Wild-type 111 99, selleck compound 120 rad59-Y92A 119 97, 124 rad59-K174A 131 111, 147 rad59-F180A 112 99, 128 rad27::LEU2 164 137, 180 rad27::LEU2 rad59-Y92A 176 136, 195 rad27::LEU2 rad59-K174A 153 126, 177 rad27::LEU2 rad59-F180A 205 183,
230 Doubling times of freshly dissected segregants were determined as described in the Methods. Displayed for each genotype is the median doubling time and 95% confidence interval, determined from at least ten independent cultures. The rad59-Y92A allele alters a conserved amino acid in another region of extensive conservation with Rad52 (Additional file 1: Figure GBA3 S1) [27, 34], and was observed to yield viable spores upon segregation with rad27::LEU2 (Figure 1). While the colonies derived from the rad27::LEU2 rad59-Y92A double mutant spores sometimes appeared smaller than the rad27::LEU2 single mutant colonies on dissection plates, neither the doubling times (p = 0.707) (Table 1; Additional file 1: Table S2), nor the ratios of G1 to S + G2/M cells (p = 0.60) (Figure 2, Additional file 1: Table S2) were significantly different for the rad27::LEU2
single and rad27::LEU2 rad59-Y92A double mutant strains. This suggests that germination of rad27::LEU2 rad59-Y92A double mutant spores may sometimes take longer than rad27::LEU2 single mutant spores. We did not observe significant effects of the tested rad59 missense alleles on doubling time (p > 0.15) (Table 1; Additional file 1: Table S2), or cell cycle distribution (p > 0.50) (Figure 2; Additional file 1: Table S2) in cells that possessed a wild-type RAD27 gene. Since all four rad59 missense mutations support steady-state levels of Rad59 that are comparable to wild-type [27], their effects on viability and growth when combined with rad27::LEU2 cannot be attributed to changes in the level of Rad59 in the cell. Altogether, these observations suggest that RAD59 plays a critical role in determining the growth characteristics of cells defective for lagging strand synthesis.