Figure 1.

K27 acetylation/deacetylation mimicking hsp82 mutants are susceptible to DNA damaging agents.A, generation of K27 and K274 acetylation/deacetylation mimicking hsp82 mutant strains by plasmid shuffling; T101Ihsp82 strain was used as a negative control for conforming the strain generation. B, serially diluted spotting of WT and hsp82 mutant strains shows no growth defect in the mutant hsp82 strains; cells were grown at two different temperatures i.e., 30 °C (left panel) and 25 °C (right panel). C, Western blot showing the stability of Hsp82 in the acetylation/deacetylation mimicking mutant hsp82 strains; Nsp1 was used as a loading control. D, quantification of two independent repeats of the above experiment shows no change in the steady state level of mutant Hsp82 proteins; the mean value (±SD) was plotted; p values were calculated using the two-tailed Student’s t test (NS, not significant). E, return to growth assay depicting the cell survivability of acetylation/deacetylation mimicking hsp82 mutant strains upon MMS treatment. Cells were treated with 0.03% and 0.05% of MMS, and percentage survivability was calculated relative to that of the untreated cells. The above experiment was conducted three independent times, and the mean value (±SD) was plotted; p values were calculated using the two-tailed Student’s t test (∗∗∗∗p < 0.0001, ∗∗∗p < 0.001 ∗∗p < 0.01, ∗p < 0.05, NS, not significant). F, percentage survivability of the hsp82 mutant strains after treatment with three different doses of UV radiations i.e., 50 J/m², 100 J/m², and 150 J/m². The mean value (±SD) of three independent repeats of the above experiment was plotted; p values were calculated using the two-tailed Student’s t test (∗∗∗p < 0.001, ∗∗p < 0.01). G, Rad51 overexpression causes partial reversal of MMS sensitivity in K27Qhsp82 and K27Rhsp82 strains; however, it showed no survival advantage once overexpressed in WT strain. The above experiment was conducted three independent times, and the mean value (±SD) was plotted; p values were calculated using the two-tailed Student’s t test (∗∗∗∗p < 0.0001, ∗∗p < 0.01, ∗p < 0.05, NS, not significant). MMS, methyl methane sulfonate.