Reversal of PCNA Ubiquitylation by Ubp10 in Saccharomyces cerevisiae
Figure 4
Increased Rev1–PCNA interaction in cells deleted for UBP10.
(A) Co-immunoprecipitation assay showing physical interaction between Rev1-myc and PCNA. PCNA was immunoprecipitated from 0.020% MMS-treated cells, blots were incubated with α-myc (to detect Rev1) or α-PCNA. The immunoblots shown are those from MMS-treated cells (a comparable result was obtained with untreated cells). As indicated the strains used in this assays were REV1-myc and REV1-myc ubp10Δ. Immunoprecipitated Rev1-myc was quantitated, normalized (to immunoprecipitated PCNA) and plotted. In (A) as well as in (C), the average and standard deviation values obtained from three independent experiments are plotted. (B) Co-immunoprecipitation assay showing physical interaction between Rev1-myc and PCNA-FLAG. PCNA-FLAG was immunoprecipitated (from protein samples crosslinked with formaldehyde, see methods) from asynchronously growing or α-factor blocked cells (as indicated), blots were incubated with α-myc (to detect Rev1) or α-FLAG (to detect PCNA). As indicated the strains used in this assays were REV1-myc POL30-FLAG and REV1-myc POL30-FLAG ubp10Δ. (C) Plots of PCNA-FLAG-co-immunoprecipitated Rev1-myc from untreated and 0.02% MMS-treated cells. Rev1-myc samples were quantitated and normalized to immunoprecipitated PCNA-FLAG. Quantitation is shown in bar diagrams. (D) Increased number of chromatin-associated Rev1 foci in MMS-treated UBP10 mutant yeast cells. Spread nuclei of wild-type and ubp10Δ strains carrying REV1-myc tagged were stained with DAPI (blue) and anti-myc antibodies (red). Cells were treated with 0.03% MMS for 1 h. The nuclei were classified in three categories according to the number of Rev1 foci. Representative ubp10Δ spread nuclei of each class and quantitation of wild-type and ubp10Δ nuclei are shown. 47 nuclei were scored for each strain.