SUPPLEMENTARY INFORMATION

Transcription

1 DOI: /ncb2579 Figure S1 Incorporation of heavy isotope-labeled amino acids and enrichment of di-glycine modified peptides. The incorporation of isotopelabeled amino acids in peptides was calculated by comparing the mislabeled fraction with the fully labeled fraction of miscleaved peptides. For calculation of lysine and arginine incorporation, miscleaved peptides containing two lysine residues or two arginine residues were used. The table shows the mean and the standard deviation of the lysine and arginine incorporation for three independent peptides for all replicate experiments performed. Fraction of peptides containing di-glycine-lysines in the enriched peptide samples. Enrichment of di-glycine modified peptides was calculated by dividing the number of quantified di-glycine modified peptides with the number of all quantified peptides. 1

2 a PAF15 ubiquitylation change 1.25 b U2OS/Strep- HA-ubiquitin UV (J/m 2 ) SILAC ratio (H/L) IR UV Pull down: Strep WCE PAF15-Ub 2 PAF15-Ub 1 PAF15-Ub 2 * PAF15-Ub 1 PAF15 PCNA-Ub 1 PCNA Chk1-pS317 c d e PAF15 DAPI sipaf15 Figure S2 UV-induced loss of PAF15 ubiquitylation of K15 and K24. Relative changes in PAF15 ubiquitylation in response to DNA damage. U2OS/Strep-HA-ubiquitin cells were subjected to SILAC-labeling in Heavy (H) or Light (L) medium, treated with IR (10 Gy) or UV (25 J/m 2 ) (SILAC Heavy) or mock-treated (SILAC Light), and harvested 1 h later. Cells were lysed under denaturing conditions, and extracts were incubated with Strep- Tactin agarose to isolate Strep-HA-ubiquitin-conjugated proteins. Bound proteins were resolved by SDS-PAGE and processed for mass spectrometry (MS)-based determination of SILAC ratios (H/L) for individual proteins. U2OS or U2OS/Strep-HA-ubiquitin cells were irradiated with indicated doses of UV, harvested 1 h later and lysed under denaturing conditions, and ubiquitylation of PAF15 was analyzed by immunoblotting of Strep-Tactin pull downs with PAF15 antibody. WCE, whole cell extract. Annotated spectrum showing mass spectrometry-based identification of K15 as a ubiquitylation site in PAF15. Annotated spectrum showing mass spectrometry-based identification of K24 as a ubiquitylation site in PAF15. PAF15 is a nuclear protein. U2OS cells were transfected with control (CTRL) or PAF15 sirnas for 48 h, fixed and immunostained with PAF15 antibody. Scale bar, 10 mm. 2

3 a b 2N 4N Exp h 6h 10h h HU release 2N N Thymidine release (h) c d U2OS/shPAF15(3 UTR)/PAF15 *PIP U2OS/ shpaf15(3 UTR)/ PAF15 *PIP PAF15 DOX sipaf15 e Pull down: Strep MW (kda) WCE U2OS/ shpaf15(3 UTR)/PAF15 K15R/K24R Strep-HA-ubiquitin UV MG132 PAF15-Ub n (WB: anti-paf15) PAF15 MCM6 WB: anti-ha High exp. PAF15-Ub 2 * PAF15-Ub 1 PAF15 Chromatin fraction MCM6 Figure S3 PAF15 K15/K24 ubiquitylation is restricted to S phase and requires PAF15 binding to PCNA. DNA content of cells in Fig. 4c was determined by flow cytometry. DNA content of cells in Fig. 4d was determined by flow cytometry. Localization of ectopic PAF15 and *PIP alleles in stable U2OS/shPAF15/PAF15 cell lines. Cells were transfected with PAF15 sirna targeting the 3 UTR (PAF15 sirna#7) for 24 h and then induced to express the transgenes by addition of Doxycycline for an additional 48 h. Cells were fixed and immunostained with PAF15 antibody. Scale bar, 10 mm. Long exposure of the PAF15 immunoblot in Fig. 4f. U2OS/shPAF15(3 UTR)/ PAF15 or K15R/K24R cell lines were transfected with PAF15 sirna targeting the 3 UTR for 24 h, induced to express ectopic PAF15 alleles by addition of Doxycycline, and transfected with Strep-HA-ubiquitin or empty vector for an additional 24 h. Cells were then synchronized in S phase by a 17-hour treatment with thymidine, released for 2 h, after which MG132 was added to the medium. Two h later, cells were exposed or not to UV as indicated, harvested 1 h later, and lysed in denaturing buffer. Ubiquitylation of PAF15 was analyzed by immunoblotting Strep-Tactin pull-downs with PAF15 antibody. MW, molecular weight. 3

4 a b Cell survival (% of CTRL) e g % cells with GFP-polη foci 100 DNA tract length (μm/min) sipaf15# #1 #5 #7 PAF15 sirna PAF15 MCM6 Camptothecin (nm) IdU, 20 min CldU, 20 min * Time after UV (h) c Cell survival (% of CTRL) d f CldU tract length (μm/min) Cell survival (% of CTRL) sipaf15 K15R K24R K15R/K24R SUPPLEMENTARY INFORMATION Povlsen et al., Figure S4 sipaf15# IR (Gy) sipaf15 sipaf15 PAF15 *PIP IdU, 20 min U2OS HU (1 h) UV (J/m 2 ) CldU, 45 min K15R/ K24R U2OS/shPAF15/PAF15 sipaf15#5 sipaf15#7 DOX Figure S4 Role of PAF15 in maintenance of genomic integrity. Immunoblot analysis of the knockdown efficiency of PAF15 sirnas used in this study. Clonogenic survival of U2OS cells treated with control or PAF15#7 sirnas and subjected to the indicated doses of Camptothecin. Results (±SEM) of three independent experiments are shown. As in (b), but with indicated doses of IR. U2OS/shPAF15(3 UTR)/PAF15 *PIP cells were transfected with control (CTRL) or PAF15#7 sirnas and left untreated or induced to express untagged PAF15 *PIP by addition of Doxycycline 24 h later. After an additional 24 h, cells were replated, then treated with the indicated doses of UV 24 h later, and survival was analyzed as above. Results (±SEM) of three independent experiments are shown. U2OS or U2OS/shPAF15/PAF15 or K15R/K24R cell lines were transfected with control (CTRL) or PAF sirnas for 24 h, induced with Doxycycline for an additional 24 h, and pulse-labeled with IdU followed by CldU for the indicated times. DNA fibre spreads were prepared as described under Experimental Procedures, and the average tract length of double-labeled (IdUCldU) fibres only was determined. At least 200 tracts were measured per experiment. Results (±SEM) of four independent experiments are shown. A summary of data obtained in this experiment is shown in Suppl. Fig. S7a. *, p<0.05. U2OS or U2OS/ shpaf15(3 UTR)/PAF15 ( or K15R/K24R) cells were transfected with CTRL or PAF15 sirnas and induced to express untagged ectopic PAF15 alleles by addition of Doxycycline (DOX) 24 h later where indicated. After an additional 24 h, cells were replated, pulse-labeled with IdU 24 h later, treated with HU for 1 h, and then washed thoroughly and labeled with CldU for 45 min. DNA fibre spreads were analyzed as in (e). Independent replicate of the experiment shown in Fig. 6c. 4

5 a No damage Cisplatin b sipaf15 sipaf15 PAF15 sipaf15 PAF15 2KR c sipaf15 sipaf15 PAF15 Figure S5 DNA fibre analysis of cells with altered PAF15 functional status. Summary of data obtained in the DNA fibre assays shown in Fig. 5e and Suppl. Fig. S4e. SEM, standard error of the mean. Representative images of DNA fibres from the experiments in Fig. 5e and 5f. DNA fibres from U2OS/shPAF15(3 UTR)/PAF15 cells treated as in Fig. 5e were fixed in methanol/acetic acid and stained with YOYO-1 (300 pm) before mounting. Representative images are shown. 5

6 Figure 3b Figure 3d Figure 3c IB: PCNA 98 IB: MCM6 Figure 3e Figure 3g IB: H2AX 64 IB: N5B Figure S6 Full gel scans of key blots in this study 6

7 Figure 4a Figure 4c IB: SET8 98 IB: MCM6 Figure 4b Figure 4d Figure S6 continued 7

8 Figure 4e Figure 4f IB: β- tubulin IB: PCNA 98 IB: MCM6 Figure 6a Figure 6d 98 Figure S6 continued 8

9 Table S1 List of all ubiquitylation sites quantified in this study. The Excel workbook contains a spreadsheet with all ubiquitylation sites (di-glycine modified lysines) quantified in this study. A detailed explanation of all columns is provided in the additional spreadsheet. Table S2 Gene Ontology annotation enrichment analysis of ubiquitylated proteins. The Excel workbook contains the results of the GO biological process (GOBP), cellular compartment (GOCC) and molecular function (GOMF) annotation enrichment analysis of ubiquitylated proteins containing sites quantified after UV irradiation. 9