Bibliografisk note

Funding Information:
This work was supported by the Francis Crick Institute (FCI receives funding from Cancer Research UK [FC001166], the UK Medical Research Council [FC001166], and the Wellcome Trust [FC001166]), and by grants to J.Q.S. from the European Research Council (ERC Agreement 693327), the Novo Nordisk Foundation (Laureate grant, NNF19OC0055875), and the Danish National Research Foundation (Chair grant, DNRF153). We thank FCI's Cell Services and Proteomics for expert technical assistance; and Svejstrup lab members for discussions. S.B. and J.Q.S. conceived the project. A.H. performed most of the experiments with contributions from S.B. (proteomics and bioinformatics for finding the Def1 homologue) J.W. (generation of human cell lines), J.C.W. (laser damage recruitment) as well as A.B.D.S and M.H.L. R.C.C, J.W.C. and J.Q.S. supervised the work. A.H. and J.Q.S. wrote the manuscript, with input from other authors. Cells were cultured in high glucose DMEM (Gibco, 41966–029) supplemented with 10% (v/v) FBS (Gibco, 10270–106), 100 U/ml penicillin, 100 μg/ml streptomycin, at 37 °C with 5% CO2 and routinely passaged 2–3 times a week. All cell lines were confirmed to be mycoplasma-free. Cell lines containing a Doxycycline inducible construct were maintained in the presence of 1 μg/ml Doxycycline (Clontech, 8634–1), refreshed every 4–6 days. Cells grown in the absence of Doxycycline were washed twice in PBS and grown for 4–5 days in DMEM supplemented with 10% (v/v) Tet-free FBS (Clontech, 631106 or Biosera, FB-1001 T/500), 100 U/ml penicillin, 100 μg/ml streptomycin. Saccharomyces cerevisiae strains used in this study were grown and manipulated using standard techniques. All strains are in the W303 background. To obtain UBAP2L and UBAP2, RNeasy Mini Kit (Qiagen, 74104) purified RNA was used to generate random hexamer primed cDNA libraries using Taqman Reverse Transcription Reagents (Thermo Fisher Scientific, N8080234). Oligos were used to amplify UBAP2L and UBAP2 and the PCR product was cloned into the TOPO vector and sequenced. Strains JSY1226 and JSY1236 were created via recombination of UBAP2L and UBAP2 cDNA into the DEF1 genomic locus of the Δdef1::URA strain (JSY568). Strains in which the URA3 marker had been replaced by the desired cDNA were selected on 5-Fluroorotic acid and correct integration was checked by PCR analysis. JSY1190 serves as a control for experiments with these strains as the WT DEF1 gene was integrated into Δdef1::URA (recreating a WT DEF1 locus). Plasmids for CRISPR knockouts were constructed as previously described [26] to insert gRNAs into plasmid PX458 to create PX458 plasmids targeting UBAP2L exon 4, UBAP2 exon 5, UBAP2L exon 2. Briefly, appropriate forward and reverse oligonucleotides were annealed and ligated into a BbsI linearized PX458 (pSpCas9(BB)−2A-GFP). See resource table for oligonucleotide sequences used. Plasmids were sequenced after cloning and transformation. Two doxycycline inducible UBAP2L expression plasmids were constructed. First the UBAP2L isoform 1 (longer) insert was amplified from UBAP2L in pcDNA5, cloned into a Zero Blunt TOPO vector (ThermoFisher Scientific) and ligated into the pTRE3G backbone with MluI and NheI. The UBAP2L isoform 2 (shorter) was constructed from the first plasmid using consecutive Q5 site directed mutagenesis (SDM) (NEB, E0554S) following manufacturer's instructions to switch the isoforms, insert a CRISPR resistant sequence over the gRNA site and convert two SNPs to their alternative forms (rs774812504 from T to C, rs17849745 from C to G). MRC5 VA TetON (WT) cells were transfected using Lipofectamine 3000 (Thermo Fisher Scientific, L3000001) following manufacturer's instructions with the relevant plasmids, as detailed below. For CRISPR knock outs: 72 h after transfection with pX458 plasmids single cells were sorted into 96 well plates with FACS and grown. Clones were expanded and checked for knock out by western blot and sequencing. For doxycycline-inducible expression: pTRE3G plasmids were co-transfected with Hygromycin linear selection marker (Takara, 31625), after transfection: 24 h later media was changed, 48 h later cells were diluted, 72 h later 200 μg/ml Hygromycin was added. Clones were expanded and checked for Doxycycline inducible UBAP2L expression by western blot. UBAP2L KO: Transfection with pX458_UBAP2L_exon4 to knock out UBAP2L. UBAP2 KO: Transfection with pX458_UBAP2_exon5 to knock out UBAP2. UBAP2/2 L KO: Co-transfection with pX458_UBAP2_exon5 and pX458_UBAP2L_exon4 to knock out UBAP2 and UBAP2L. DKO: Co-transfection with pX458_UBAP2_exon5 and pX458_UBAP2L_exon4 to knock out UBAP2 and UBAP2L, followed by transfection with pTRE3G_UBAP2L_isoform1 to allow Doxycycline-inducible expression of UBAP2L isoform 1. These cells were maintained in the presence of Doxycycline-induced UBAP2L (DKO + UBAP2L). Cells were used for experiments as DKO 4–5 days after Dox withdrawal when UBAP2L expression was at background levels. DKO clF2 and DKO clH2: Transfection with pTRE3G_UBAP2L_isoform2_CRISPR_resistant to allow Doxycycline-inducible expression of CRISPR-resistant UBAP2L isoform 2, followed by co-transfection with pX458_UBAP2_exon5 and pX458_UBAP2L_exon2 to knock out UBAP2 and UBAP2L. These cells were maintained in the presence of Doxycycline-induced UBAP2L. Cells were used for experiments as DKO 4–5 days after Dox withdrawal when UBAP2L expression was at background levels. UV-irradiation was performed as previously described [37]. Media was removed, cells were irradiated with either Stratalinker (Stratagene) or a custom-made UV conveyor belt and the same media replaced. UVC was used and doses were monitored using a UV meter (Progen Scientific) in all experiments. Cells were plated in 15 cm plates to be around 80% confluent the following day when they were UV irradiated at 30 J/m2. At time points indicated cells were washed with ice cold PBS, scraped into an Eppendorf tube in ice cold PBS and pelleted at 300 g, 4 °C, 5 min. Cellular fractionation was carried out as previously described [13]. All buffers contained cOmplete protease inhibitor (Sigma, 5056489001) and phosSTOP phosphatase inhibitor (Sigma, 4906837001). Pellets were resuspended in 500 μl hypotonic buffer (10 mM HEPES pH 7.5, 10 mM KCl, 1.5 mM MgCl2) and incubated on ice for 15 min, homogenized with 20 strokes using a loose pestle and spun at 3,000 g, 4 °C, 15 min. Supernatant was taken as the cytoplasmic extract and corrected to 10% (v/v) glycerol, 3 mM EDTA, 0.05% (v/v) NP-40, 150 mM NaCl. The remaining nuclear pellets were resuspended in 500 μl nucleoplasmic extraction buffer (20 mM HEPES pH 7.5, 1.5 mM MgCl2, 150 mM Potassium Acetate, 10% v/v Glycerol, 0,05% (v/v) NP-40) and incubated on ice for 20 min, then spun at 20,000 g, 4 °C, 20 min to pellet chromatin. Supernatant was taken as the Nucleoplasmic fraction. The remaining chromatin pellets were resuspended in 200 μl chromatin digestion buffer (20 mM HEPES pH 7.9, 1.5 mM MgCl2, 150 mM NaCl, 10% (v/v) Glycerol, 0.05% (v/v) NP-40, 1:1000 BaseMuncher (Abcam, ab270049)) and incubated for 1 h, 4 °C, rotating, then spun at 20,000 g, 4 °C, 20 min, supernatant was taken as the low salt chromatin fraction. Pellets were resuspended in 120 μl high salt chromatin extraction buffer (20 mM HEPES pH 7.9, 500 mM NaCl, 3 mM EDTA, 1.5 mM MgCl2, 10% v/v Glycerol, 0.05% (v/v) NP-40) and incubated on ice for 20 min. 280 μl high salt dilution buffer (20 mM HEPES pH 7.9, 3 mM EDTA, 1.5 mM MgCl2, 10% (v/v) Glycerol, 0.05% (v/v) NP-40) was added and samples spun at 20,000 g, 4 °C, 15 min. Supernatant was pooled with low salt chromatin fraction to form the chromatin fraction. Protein concentration was measured (Protein Assay Dye Reagent, Bio-Rad, 5000006) in a plate reader. Whole cell extracts were collected from 6 well plates by washing with PBS, adding 100 μl RIPA buffer (TrisHCL pH 7.5 50 mM, NaCl 150 mM, NP40 1% (v/v), Sodium Deoxycholate 0.5% (w/v), SDS 0.1%) containing cOmplete protease inhibitor (Sigma, 5056489001) and phosSTOP phosphatase inhibitor (Sigma, 4906837001), incubating on ice for 2 min and then scraping to Eppendorf tubes. Samples were sonicated in a Bioruptor water bath sonicator (Diagenode) on high 30 s ON/ 30 s OFF for 5 min. Samples were then spun at 20,000 g, 4 °C, 5 min and supernatant taken. Protein concentration was measured (Protein Assay Dye Reagent, Bio-Rad, 5000006) in a plate reader. 10 or 15 μg protein/lane was separated on 4–15% Criterion TGX (Bio-Rad, 5671084) or NuPAGE 3 to 8% Tris-Acetate (Thermo Fisher Scientific, WG1602) gels and transferred to nitrocellulose (GE Healthcare Life Sciences, 10600002). Membranes were blocked in 5% (w/v) skimmed milk in PBS-T (PBS, 0.2% (v/v) Tween20) for 1 h at room temperature and incubated in primary antibody in 5% (w/v) skimmed milk in PBS-T overnight at 4 °C. Membranes were washed 3 times in PBS-T, incubated for 1 h at room temperature in HRP-conjugated secondary antibodies (anti-mouse, Santa Cruz, sc516102 or anti-rabbit, Jackson, 711035152) and visualized with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, 34580). Supplementary Figs. S1B and S3C used Immobilon-FL membrane (Merck, IPFL00010), were blocked in Intercept blocking buffer (Licor, 927–70001), fluorescent secondary antibodies (Invitrogen, anti-mouse Alexa Fluor 680, A10038, anti-rabbit Alexa Fluor 680 or anti-rabbit Alexa Fluor Plus 800) and were visualized on LiCor Odyssey CLx. Cells were seeded in 6-well plates (500 cells/well for untreated, 2,000–5,000 cells/well for UV treated). The following day media was removed and cells irradiated with varying UV doses and the same media replaced. Colonies were allowed to grow for 2 weeks before being washed carefully with PBS and fixed with 3.7% Formaldehyde for 20 m and washed with water. Colonies were stained with 0.1% (w/v) crystal violet solution, scanned and colonies counted manually. GST-Dsk2 affinity resin was prepared as previously described [37]. One Shot BL21 (DE3) Star bacteria were transformed with pGEX3-Dsk2 plasmid according to manufacturer's instructions. 10 ml overnight culture was used to inoculate a 250 ml culture grown to OD600 = 0.6, all at 37 °C in LB with 100 μg/ml Ampicillin (VWR, 171254–25) and shaking. Expression was induced with 1 mM IPTG and bacteria grown at 30 °C with shaking for 4 h, cells were then pelleted and snap frozen in liquid nitrogen. The pellet was resuspended in 40 ml PBS with protease inhibitors (2.2 mM PMSF, 2 mM Benzamidine HCL, 2 μM Leupeptin, 1 μg/ml Pep statin A) and sonicated (Branson Digital Sonifer 250) at 30% output for 15 s ON/30 s OFF pulses for a total of 10 min ON time on ice. Triton X100 was added to 0.5%, mixed gently and incubated on ice for 30 min. Following a 12,000 g, 4 °C, 10 min spin the supernatant (lysate) was taken and DTT added to 2 mM final concentration. 1 ml Glutathione Sepharose 4B Beads (Sigma, GE17–0756-01) were spun at 700 g, washed twice in PBS, added to the lysate and incubated at 4 °C with rotation for 4 h. Beads were then spun at 700 g, washed for 5 min twice with ice cold PBS + 0.1% Triton X100 then once with ice cold PBS. Finally 1 ml GST-DSK2 affinity resin was resuspended in 3 ml PBS and stored at 4 °C before use. Cells were plated in 1×10 cm dish per condition such that cells were around 80% confluent the following day for UV-irradiation. Following UV-irradiation, at the indicated times media was removed, cells washed with PBS alone or PBS containing 2 mM NEM (200 mM stock in ethanol made fresh). 800 μl TENT buffer (50 mM TrisHCL pH7.4, 2 mM EDTA, 150 mM NaCl, 1% Triton X100) containing 2 mM NEM (200 mM stock in ethanol made fresh) and protease inhibitors (2.2 mM PMSF, 2 mM Benzamidine HCL, 2 μM Leupeptin, 1 μg/ml Pep statin A) was added and incubated for 5 min, then scraped into an Eppendorf or falcon. Samples were incubated on ice for 20 min then sonicated, Branson Digital Sonifer 250 at 20% output for 10 s or Bioruptor water bath sonicator (Diagenode) on high 30 s ON/ 30 s OFF for 7 min. MgCl2 to 3 mM and BaseMuncher 1:1,000 (Expedeon, BM0100) were added to each sample and incubated for 1 h at 4 °C with rotation. Samples were spun at 20,000 g, 4 °C, 5 min and the supernatant taken. Protein concentration was measured (Protein Assay Dye Reagent, Bio-Rad, 5000006) and each sample adjusted with TENT buffer to 750 μl at 1 mg/ml, a 1% input was taken and boiled with Sample buffer. 120 μl per sample of bead suspension (30 μl packed bead volume, GST-DSK2 affinity resin) was spun down at 700 g and resuspended in equivalent volume of TENT buffer. 120 μl was added to each sample and rotated overnight at 4 °C. Beads were spun down at 700 g, washed with 5 min, 4 °C, rotating incubations twice in TENT buffer and once in PBS. 50 μl 1x Sample Buffer was added and the sample boiled for 2 min. 1% input and 20% sample were run on a 4–15% Criterion TGX gel (Bio-Rad, 5671084) and normal Western blotting procedure followed. Cells were plated in MatTek dishes (35 mm, No. 2 14 mm diameter glass) at a density of 2 × 106 per dish in media containing 200 µg/ml G418. Cells were transfected with 400 ng of either Halo-Elongin A or mCherry-CUL5 plasmid DNA [41] using FugeneHD (Promega), 24 h prior to imaging. To label Halo-tagged Elongin A with rhodamine 110 in living cells, HaloTag ®R110Direct™ ligand was added to a final concentration of 100 nM, and cells were allowed to incubate overnight without washing as directed in the manufacturer's protocol. 30 min prior to imaging, culture medium was replaced with phenol-red free medium containing the same additives plus 1 µg/ml Hoechst 33258 to label nuclei and sensitize cells to UV-irradiation. UV-microirradiation was performed by subjecting cell nuclei to laser micro-irradiation in a 200 × 3 pixel (34 × 0.51 µm) stripe. Micro-irradiation was performed with 100% 405 nm laser power and cells were exposed to 500–700 µW for approximately 3 s (40 iterations). Normal cell and nuclear morphology were preserved over the time scale of the experiment. Micro-irradiation and imaging were performed on a Perkin Elmer UltraVIEW VoX spinning disk microscope, which included a Yokagawa CSU-X11 spinning disk, an ORCA-R2 camera (Hamamatsu), and a Perkin Elmer PhotoKinesis accessory. The microscope base was a Carl Zeiss Axiovert 200 M equipped with a 40 × 1.3 NA Plan-Apochromat objective and a 37 °C, 5% CO2 incubator (Solent Scientific). R110Direct labeled Halo-ELOA was excited with the 488 nm laser and imaged with a 500–550 emission filter. mCherry was excited with the 561-nm laser and imaged through a 415–475-nm, 580–650-nm multiband emission filter. Hoechst was excited with the 405-nm laser and imaged through a 415–475-nm, 580–650-nm multiband emission filter. Laser power and exposure time were adjusted before-hand to maximize image quality and minimize photobleaching; absence of significant photobleaching was confirmed by observing unperturbed cells in the acquisition field of view. Overnight yeast cultures were diluted to early logarithmic phase and grown for approximately 4 h. Tenfold serial dilutions were made and spotted on YPD agar plates and incubated at 30 °C for 2–3 days. After growth, the plates were photographed using a GelDoc XR (Bio-Rad). Details of statistical tests used and numbers of replicates are detailed in the relevant figure legend. Throughout * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Statistical analysis was carried out in Prism 7 software. Quantification of western blots was carried out in ImageStudioLite software.

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