Fig. 8: CRL4CSA and proteasome-mediated removal of DPCs.
From: Transcription-coupled DNA–protein crosslink repair by CSB and CRL4CSA-mediated degradation
a, Relative immobile fractions of non-replicating mScarlet-I–CSB FRAP in cells arrested following treatment with 1 µM palbociclib for 24 h. Cells were mock treated or treated with 50 µM proteasome inhibitor (MG132), 10 µM VCP inhibitor (VCPi; NMS873) or 20 µM neddylation inhibitor (NAEi; MLN4924) before treatment with 1 mM FA for 30 min. Values represent the mean ± s.e.m. n (left to right) = 25, 24, 34, 26, 24, 30, 40, 31, 27, 30, 39, 34, 27, 30, 39, 34, 27, 30, 40 and 33 cells from 3 (NAEi) or 4 (VCPi) independent experiments. Unpaired two-tailed t-test. b, Top: scheme of experiment. Bottom: IP of pSer2-modified elongating Pol II followed by immunoblotting with the indicated antibodies either directly after a 30 min FA pulse (1 mM) or after recovery for the indicated time. This experiment was performed twice with similar results. Untr., untreated. c, Relative immobile fractions of non-replicating mScarlet-I–CSB FRAP in siRNA-transfected cells, which were arrested in G1 with 1 μM palbociclib for 24 h before treatment with 1 mM FA for 30 min. Graphs represent the mean ± s.e.m. n (left to right) = 36, 28, 38, 24, 35, 28, 36, 28, 29 and 28 cells from 3 independent experiments. Unpaired two-tailed t-test. d, Quantification of recovery of transcription in RPE1 cells transfected with the indicated siRNA. Cells were treated with a 30 min FA (1 mM) pulse and left to recover for the indicated times, including a 30 min pulse labelling with EU. RFI of EU were normalized to untreated levels and set to 100%. Black lines indicate the average integrated density ± s.e.m. n (left to right) = 688, 652, 331, 633, 611, 466, 726, 391, 265, 721, 418 and 313 cells from 2 (siCSB) or 3 (siCtrl and siDDB1) independent experiments. Unpaired two-tailed t-test. e, Relative clonogenic survival assay in WT (MRC-5 sv40), CS-A (CS3BE sv40), CS-B (CS1AN sv40) and UVSS-A (TA-24 sv40) cells with the indicated doses of FA for a 1 h pulse. Graphs were normalized to the untreated colony number, which was set at 100%. Graphs represent the mean ± s.e.m. from five independent experiments. Unpaired two-tailed t-test. f, TC-DPC repair model. TC-DPC repair is initiated when DPC-stalled Pol II is recognized by CSB, which recruits CRL4CSA E3 ligase. UVSSA stabilizes CSB through USP7. Ubiquitin (Ub)-DPC ubiquitylation by CRL4CSA drives VCP-dependent and proteasome-dependent DPC degradation followed by transcription restart. Functional TC-DPC repair may explain the phenotypic differences in the TC-NER syndromes CS and UV-sensitive syndrome. Source numerical data (a,c–e) and unprocessed blots (b) are available in the source data. Scheme in f was created using BioRender (https://biorender.com).
