van der Weegen Y , Golan-Berman H , Mevissen TET , Apelt K , González-Prieto R , Goedhart J , Heilbrun EE , Vertegaal ACO , van den Heuvel D , Walter JC , Adar S , Luijsterburg MS .

Howard Hughes Medical Institute , R01 HL098316 NHLBI NIH HHS

	Fig. 1: CSA is recruited to DNA damage-stalled RNAPIIo by CSB. a Outline of a new IP method to isolate RNAPIIo and associated proteins from mock-treated or UV-irradiated (20 J/m2) U2OS (FRT) cells. b Endogenous RNAPII Co-IPs on WT cells stained for the indicated TCR proteins. Note that it is not possible to stain for all these proteins on one membrane. This panel is a composite of several representative Co-IPs. See Supplementary Fig. 1a for each individual Co-IP. c Endogenous RNAPII Co-IP followed by slot blot analysis of CPDs d Western blot analysis of CSA, CSB, and UVSSA knockout cells complemented with inducible GFP-tagged versions of these proteins (n = 2). See Supplementary Fig. 2a, b for validation of knockouts by sequencing. e Clonogenic Illudin S survival of WT, CSA, CSB, and UVSSA knockout and rescue cell lines. Each symbol represents the mean of an independent experiment (n = 2 for all except for WT in UVSSA-KO figure which is n = 3) each experiment contains two or three technical replicates. Endogenous RNAPII Co-IP on f WT, CSA, CSB, and UVSSA knockout cells, g CSB-KO stably expressing GFP-CSB, and h WT, XPC, CSA, CSB, and UVSSA knockout cells. The asterisk in e indicates the heavy chain of the RNAPII antibody. At least two independent replicates of each IP experiment were performed obtaining similar results. Source data are provided as a Source Data file.
	Fig. 2: CSA interacts with the newly identified C-terminal CIM of CSB. a Outline of the chromatin-tethering approach in U2OS 2-6-3 cells. b A schematic representation of CSB and its deletion mutants. c Recruitment of CSA-GFP to the LacO array upon tethering of the indicated mCherry-LacR fusion proteins (scale bar = 5 µm). See Supplementary Figs. 3 and 4 for a full overview of all tested mutants. d Quantification of CSA-GFP and mCherry-LacR-CSB co-localization at the LacO array. Each symbol represents the mean of an independent experiment (n = 2 for all except LacR-NLS and LacR-CSBWT which is n = 8, >50 cells collected per experiment). e Sequence alignment of CSB orthologues. See Supplementary Fig. 5 for additional alignments. Source data are provided as a Source Data file.
	Fig. 3: The CIM of CSB mediates the recruitment of CSA to DNA damage-stalled RNAPIIo. a A schematic representation of CSB and the CSBΔCIM mutant. b Western blot analysis of U2OS (FRT) and CSB-KO complemented with either GFP-CSBWT or GFP-CSBΔCIM (n = 2). c Co-IP of GFP-CSBWT and GFP-CSBΔCIM on the combined soluble and chromatin fraction. d Endogenous RNAPII Co-IP in GFP-CSBWT and GFP-CSBΔCIM cell lines. See also Supplementary Fig. 6e for additional Co-IP data. e Clonogenic Illudin S survival of WT and CSB-KO cell lines and the GFP-tagged CSB rescue cell lines. Each symbol represents the mean of an independent experiment (n = 2), each of which is based on two technical replicates. Note that the same survival data for WT, CSB-KO and CSB-KO + GFP-CSB is also shown in Fig. 1e. f In vitro ubiquitylation of recombinant Xenopus laevis (xl) and Homo sapiens (hs) CSB variants with recombinant xlCRL4CSA, E1, E2, ubiquitin, and ATP. At indicated times, in vitro ubiquitylation reactions were stopped and blotted with anti-FLAG (top three panels) or anti-xlCSA (bottom panel) antibodies. See also Supplementary Fig. 6d. g Immobilized recombinant CSB variants were incubated with Xenopus laevis nucleoplasmic extract (NPE), recovered, and blotted with anti-FLAG (top panel) or anti-xlCSA (bottom panel) antibody. At least two independent replicates of each IP and in vitro ubiquitylation experiment were performed obtaining similar results. Source data are provided as a Source Data file.
	Fig. 4: UVSSA is recruited to DNA damage-stalled RNAPIIo by CSA a Western blot analysis of UVSSA-KO, UVSSA/CSA-dKO, and UVSSA/CSB-dKO complemented with GFP-UVSSA (n = 2). b Clonogenic Illudin S survival of WT, UVSSA-KO, UVSSA/CSA-dKO, and UVSSA/CSB-dKO cell lines complemented with GFP-UVSSA. Each symbol represents the mean of an independent experiment (n = 2 for all except for UVSSA-KO which is n = 3), each of which is based on two technical replicates. c Endogenous RNAPII Co-IP on UVSSA-KO, UVSSA/CSA-dKO, and UVSSA/CSB-dKO complemented with GFP-UVSSA. d Co-IP of GFP-UVSSA in UVSSA-KO, UVSSA/CSA-dKO, and UVSSA/CSB-dKO cell lines. The asterisk in c indicates the heavy chain of the RNAPII antibody. At least two independent replicates of each IP experiment were performed obtaining similar results. Source data are provided as a Source Data file.
	Fig. 5: CSA, CSB, and UVSSA are equally important for TFIIH recruitment.a Endogenous RNAPII Co-IP in UVSSA-KO, UVSSA/CSA-dKO, and UVSSA/CSB-dKO complemented with GFP-UVSSA. b Volcano plot depicting the statistical differences of the MS analysis on GFP-UVSSA pull-down in mock-treated and UV-irradiated samples. The enrichment (log2) is plotted on the x-axis and the significance (t-test -log p value) is plotted on the y-axis. All significantly UV-induced hits are indicated in green. Several selected hits are shown in red (Link to the interactive volcano plots: GFP-UVSSA vs GFP-UVSSA + UV). c Co-IP of GFP-UVSSA in UVSSA-KO and UVSSA-dKO cells complemented with GFP-UVSSA. d Endogenous RNAPII Co-IP in WT, CSB-KO, CSA-KO, UVSSA-KO, and XPA-KO cells. e Endogenous RNAPII Co-IP in WT and UVSSA-KO cells and CSA-KO, CSB-KO, and UVSSA-KO cells complemented with GFP-tagged versions of these proteins. The asterisk in d and e indicates the heavy chain of the RNAPII antibody. f CPD XR-seq repair signal 3 Kb upstream and 5 Kb downstream of the annotated TSS of 16.088 genes in WT and UVSSA-KO cells. Signal is plotted separately for the transcribed (red) and non-transcribed (black) strands. The data represent the average of two independent experiments with a bin size of 40 nt. See also Supplementary Fig. 7a for additional XR-seq data. At least two independent replicates of each IP experiment were performed obtaining similar results. Source data are provided as a Source Data file.
	Fig. 6: UVSSA is the key protein that recruits TFIIH. a A schematic representation of UVSSA WT and deletion mutants. The CSA-interacting region (CIR) and TFIIH-interacting region (TIR) are indicated. b Recruitment of CSA-GFP and TFIIH (p89) to the LacO array upon tethering of the indicated mCherry-LacR fusion proteins (scale bar = 5 µm). c Quantification of CSA-GFP and endogenous TFIIH (p89) co-localization at the LacO array. Each symbol represents the mean of an independent experiment (n = 2, >50 cells collected per experiment). d Western blot analysis of U2OS (FRT) and UVSSA-KO cells complemented with GFP-UVSSAWT, GFP-UVSSAΔCIR, and GFP-UVSSAΔTIR (n = 2). e Co-IP of GFP-UVSSAWT, GFP-UVSSAΔCIR, and GFP-UVSSAΔTIR. f Endogenous RNAPIIo Co-IP in GFP-UVSSAWT, GFP-UVSSAΔCIR, and GFP-UVSSAΔTIR cell lines. At least two independent replicates of each IP experiment were performed obtaining similar results. See also Supplementary Fig. 7b, c for additional Co-IP data. Source data are provided as a Source Data file. interacting region (CIR) and TFIIH-interacting region (TIR) are indicated. b Recruitment of CSA-GFP and TFIIH (p89) to the LacO array upon tethering of the indicated mCherry-LacR fusion proteins (scale bar = 5 µm). c Quantification of CSA-GFP and endogenous TFIIH (p89) co-localization at the LacO array. Each symbol represents the mean of an independent experiment (n = 2, >50 cells collected per experiment). d Western blot analysis of U2OS (FRT) and UVSSA-KO cells complemented with GFP-UVSSAWT, GFP-UVSSAΔCIR, and GFP-UVSSAΔTIR (n = 2). e Co-IP of GFP-UVSSAWT, GFP-UVSSAΔCIR, and GFP-UVSSAΔTIR. f Endogenous RNAPIIo Co-IP in GFP-UVSSAWT, GFP-UVSSAΔCIR, and GFP-UVSSAΔTIR cell lines. At least two independent replicates of each IP experiment were performed obtaining similar results. See also Supplementary Fig. 7b, c for additional Co-IP data. Source data are provided as a Source Data file.
	Fig. 7: The CIR and TIR of UVSSA are crucial for TCR. a Clonogenic Illudin S survival of WT and UVSSA-KO cell lines and the GFP-tagged UVSSA rescue cell lines. Each symbol represents the mean of an independent experiment (n = 3 for all except for GFP-UVSSAΔCIR which is n = 2), each of which is based on two technical replicates. Note that the same survival data for UVSSA-KO is also shown in Fig. 4b. b CPD XR-seq repair signal 3 Kb upstream and 5 Kb downstream of the annotated TSS of 16,088 genes in GFP-UVSSAWT, GFP-UVSSAΔCIR, and GFP-UVSSAΔTIR. Signal is plotted separately for the transcribed (red) and non-transcribed (black) strands. The data represent the average of two independent experiments for GFP-UVSSAWT and one single experiment for GFP-UVSSAΔCIR and GFP-UVSSAΔTIR with a bin size of 53 nt. See also Supplementary Fig. 9 for additional XR-seq data. c Model of how the assembly of CSB, CSA, and UVSSA targets the TFIIH complex to DNA damage-stalled RNAPIIo. Source data are provided as a Source Data file.

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