FIGURE 4.

Overexpression of Rad52 rescues the lethality of dna2-K1080E, but it still displays growth defects. A and B, the survival (A) and growth (B) rates of wild type (WT+Rad52) and dna2-K1080E (dna2-KE+Rad52) cells overexpressing Rad52 were measured as described under “Materials and Methods.” The experiment was repeated three times, and the results are presented with error bars. C, the levels of H2A-Ser¹²⁹ phosphorylation were examined. The logarithmically growing cells were treated with α-factor (3 μg/ml) followed by incubation at 25 °C for 4 h. The cells were withdrawn at the indicated time point, and the total cellular proteins were prepared as described under “Materials and Methods” followed by immunoblotting with α-H2A-Ser129P antibodies raised against phosphorylated serine 129 residue of H2A (ab17353, Abcam). Cells grown for 1 h in the presence of 0.033% methyl methanesulfonate were used as a positive control for the detection of phosphorylated H2A-Ser¹²⁹. Histone H3 was used as loading control. α-H3, antibodies against histone H3. D, the relative distribution of the G₁-, S-, and G₂/M-phase cells in logarithmically growing cells was determined using microscopic analyses. Cells were categorized into three groups by their budding states: G₁, unbudded cells; S, budding cells; G₂/M, dumbbell-shaped cells. The experiment was repeated three times, and the results are presented with error bars. E, the morphology of nuclei was examined microscopically using cells stained with 4′,6-diamidino-2-phenylindole. The population of G₂/M-phase cells with normal (two nuclei, segregated) and defective nuclear segregation (one nucleus, unsegregated) was determined. The experiment was repeated three times, and the results are presented with error bars.