Figure 3.

Hda1 deletion is epistatic to the K27 acetylation/deacetylation mimicking Hsp82 mutants.A, return to growth assay depicting the cell survivability of hda1Δ strain upon 0.03% MMS and 0.05% MMS. The 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 < 0.001, ∗∗p < 0.01). B, percentage survivability of hda1Δ strain after treatment with 50 J/m², 100 J/m² and 150 J/m² of UV doses. The mean value (±SD) of three independent repeats of the above experiments was plotted; p values were calculated using the two-tailed Student’s t test (∗∗∗p < 0.001). C, percentage survivability of K27Qhsp82hda1Δ strain with respect to K27Qhsp82 strain upon 0.03% MMS and 0.05% MMS treatment. 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 (NS, not significant). D, percentage survivability of K27Qhsp82hda1Δ strain after treatment with 50 J/m², 100 J/m², and 150 J/m² of UV doses. The mean value (±SD) of three independent repeats were plotted; p values were calculated using the two-tailed Student’s t test (∗∗∗p < 0.001). E and F, percentage survivability of K27Rhsp82hda1Δ strain showed similar dose-dependent sensitivity to K27Rhsp82 strain upon exposure to various doses of MMS and UV radiations. The above experiments were conducted three independent times, and the mean value (±SD) was plotted; p values were calculated using the two-tailed Student’s t test (∗∗p < 0.01, NS, not significant). G, Aha1 protein was immunoprecipitated with Aha1 specific antibody, from WT, hda1Δ, rpd3Δ, and hda1Δrpd3Δ strains, the pellet was probed for the presence of Hsp82 in each fraction. The expression of Aha1 and Hsp82 were probed in each strain and presented in the bottom panel as lysate. H, Western blots showing CoIP of Rad51 with Hsp82 in WT and hda1Δ strains. I, the above experiment was repeated two times and the quantification of Western blots showed a moderate reduction in interaction of Rad51 with Hsp82 in the absence of Hda1; the mean value (±SD) was plotted; p values were calculated using the two-tailed Student’s t test (∗∗∗p < 0.001). J, Western blot showing the stability of Rad51 in hda1Δ and rpd3Δ strains. K, Western blot showing the endogenous level of Rad51 in hda1Δ strain in presence and absence of proteasome-inhibitor MG132. L, Western blot showing the endogenous level of Rad51 in rpd3Δ strain in presence and absence of proteasome-inhibitor MG132. CoIP, coimmunoprecipitation; MMS, methyl methane sulfonate.