Cells lacking UBP10 accumulate mono- and di-ubiquitylated PCNA in response to DNA damage and replicative stress.

(A) A polyclonal rabbit antibody that specifically detects PCNA forms in yeast cell extracts. Immunoblot analysis with (affinity purified) rabbit α-PCNA antibody of TCA-protein extracts from wild-type, rad18Δ (unable to ubiquitylate PCNA), pol30^K164R (unable to ubiquitylate or SUMOylate PCNA), mms2Δ (unable to biubiquitylate PCNA) and siz1Δ (unable to SUMOylate PCNA) cells treated 90 minutes with 0.020% MMS and resolved in 10% or 12% polyacrylamide gels (as indicated), note that right lane of the 10% gel correspond to wild-type cells treated with 0.3% MMS (conditions where only SUMOylated PCNA forms are detected). (B) Di-ubiquitylated PCNA accumulation in MMS-treated single ubp1 to ubp17 deletions in S.cerevisiae. Graph of di-ubiquitylated PCNA accumulation in 0.020% MMS-treated single UBP1-17 deletions in S.cerevisiae. Wild-type and single mutant cells exponentially grown at 30°C were treated 60 minutes with 0.020% MMS. TCA-cell extracts were analyzed for PCNA ubiquitylation by Western blot, quantitated and plotted. Average values from three independent assays are plotted. (C) Immunodetection of ubiquitylated forms of PCNA in wild-type, ubp10Δ, pol30^K164R and ubp10Δ pol30^K164R TCA-cell extracts to show that UBP10 mutant cells accumulate K164 mono-ub and di-ubPCNA forms. Immunodetection of mono-ubiquitylated (D) and di-ubiquitylated PCNA (E) in wild-type and ubp10Δ cells treated with 0.020% MMS, 200 mM HU, 0.2 µg/ml 4-NQO and 100 J/m² UV-light (as indicated). Rad53 phosphorylation was used to test checkpoint activation upon treatments.