KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Rabbit polyclonal anti-XPC CT Antigen: X. laevis XPC 1049–1062 | This study | N/A |
| Rabbit polyclonal anti-XPC NT Antigen: X. laevis XPC 1–20 | This study | N/A |
| Rabbit polyclonal anti-CSB Antigen: X. laevis CSB 1357–1370 | This study | N/A |
| Rabbit polyclonal anti-CSA Antigen: X. laevis CSA 380–399 | van der Weegen et al.15 | N/A |
| Rabbit polyclonal anti-UVSSA Antigen: X. laevis UVSSA 718–737 | This study | N/A |
| Rabbit polyclonal anti-STK19 Antigen: X. laevis STK19 1–15 (C9S) | This study | N/A |
| Rabbit polyclonal anti-RPB1 Antigen: C(YSPTSPS)4 | This study | N/A |
| Rabbit polyclonal anti-XPD Antigen: X. laevis XPD 741–760 | This study | N/A |
| Rabbit polyclonal anti-ELOF1 Antigen: X. laevis ELOF1 full-length | This study | N/A |
| Rat monoclonal anti-RPB1 phospho-Ser5 (clone 3E8) | Kind gift from Stephen Buratowski | RRID:AB_2687451 |
| Rat monoclonal anti-RPB1 phospho-Ser2 (clone 3E10) | Kind gift from Stephen Buratowski | RRID: AB_2784639 |
| Rabbit polyclonal anti-TDG | Slenn et al.47 | N/A |
| Rabbit polyclonal anti-H3 | Cell Signaling Technology | Cat# 9715; RRID:AB 331563 |
| Rabbit polyclonal anti-p97 | Heubes et al.49 | N/A |
| Mouse Anti-Rabbit IgG (H+L), unconjugated | Jackson ImmunoResearch | Cat# 211–005-109; RRID:AB 2339147 |
| Mouse Anti-Rabbit IgG (L), HRP-conjugated | Jackson ImmunoResearch | Cat# 211–032-171; RRID:AB 2339149 |
| Rabbit Anti-Mouse IgG (H+L), HRP-conjugated | Jackson ImmunoResearch | Cat# 315–035-003; RRID:AB 2340061 |
| Goat Anti-Rat IgG (H+L), HRP-conjugated | Thermo Fisher Scientific | Cat# 31470 RRID:AB 228356 |
| Bacterial and virus strains | ||
| E. coli Rosetta 2(DE3)pLacI | Sigma-Aldrich | Cat# 71404–3 |
| E. coli OverExpress C41(DE3) | Sigma-Aldrich | Cat# CMC0017 |
| E. coli DH5α | NEB | Cat# C2987H |
| E. coli DH10EMBacY | Geneva Biotech | N/A |
| E. coli BL21-CodonPlus (DE3)-RIL | Agilent | Cat# 230245 |
| Biological samples | ||
| Sus scrofa thymus | Pel-Freez | Cat# 59451 |
| Chemicals, peptides, and recombinant proteins | ||
| BamHI-HF | NEB | Cat# R3136M |
| SacI-HF | NEB | Cat# R3156L |
| BbsI-HF | NEB | Cat# R3539L |
| PmlI | NFR | Cat# R0532S |
| EcoRI-HF | NEB | Cat# R3101S |
| KpnI-HF | NEB | Cat# R3142S |
| XhoI | NEB | Cat# R0146S |
| AflII | NEB | Cat# R0520S |
| I-CeuI | NEB | Cat# R0699S |
| NEBuilder HiFi DNA Assembly Master Mix | NEB | Cat# E2621L |
| Q5 Hot Start High-Fidelity 2X Master Mix | NEB | Cat# M0494L |
| QIAprep Spin Miniprep Kit | Qiagen | Cat# 27106 |
| QIAquick Gel Extraction Kit | Qiagen | Cat# 28706 |
| AMPure XP Reagent | Beckman | Cat# A63881 |
| RNAClean XP | Beckman | Cat# A63987 |
| cis-Diammineplatinum(II) dichloride | Sigma-Aldrich | Cat# P4394 |
| ATP | Sigma-Aldrich | Cat# A5394 |
| Phosphocreatine | Sigma-Aldrich | Cat# P6502 |
| Creatine Phosphokinase | Sigma-Aldrich | Cat# C3755 |
| DTT | Bio-Rad | Cat# 1610611 |
| Nocodazole | Sigma-Aldrich | Cat# M1404 |
| α-amanitin | Santa Cruz | Cat# sc-202440 |
| Recombinant RNasin Ribonuclease Inhibitor | Promega | Cat# N2511 |
| MLN4924 | Active Biochem | Cat# A-1139 |
| Proteinase K | Roche | Cat# 3115879001 |
| [α-32P]UTP | Revvity | Cat# BLU507H250UC |
| [α-32P]dCTP | Revvity | Cat# BLU013H500UC |
| 30% w/v Polyethylene glycol 20,000 | Hampton Research | Cat# HR2–609 |
| Xylene Cyanol FF | Sigma-Aldrich | Cat# X4126 |
| Bromophenol Blue | Sigma-Aldrich | Cat# B0126 |
| RNase A | Sigma-Aldrich | Cat# R5503 |
| SYBR Gold Nucleic Acid Gel Stain | Invitrogen | Cat# S11494 |
| Precision Plus Protein Dual Xtra Prestained Protein Standards | Bio-Rad | Cat# 1610377 |
| AminoLink Coupling Resin | Thermo Fisher Scientific | Cat# 20382 |
| rProtein A Sepharose Fast Flow | Cytiva | Cat# 17127903 |
| Dynabeads Protein A | Invitrogen | Cat# 10001D |
| Protein A Mag Sepharose Xtra | Cytiva | Cat# 28967062 |
| BSA | Fisher Scientific | Cat# BP1600–100 |
| InstantBlue Protein Stain | Novus | Cat# ISB1L |
| One-Step Blue Protein Gel Stain | Biotium | Cat# 21003 |
| SuperSignal West Dura Extended Duration Substrate | Thermo Fisher Scientific | Cat# 34075 |
| complete EDTA-free Protease Inhibitor Cocktail | Roche | Cat# 11873580001 |
| ANTI-FLAG M2 Affinity Gel | Sigma-Aldrich | Cat# A2220 |
| 3x FLAG Peptide | Sigma-Aldrich | Cat# F4799 |
| Glutathione Sepharose 4B | Cytiva | Cat# GE17–0756-01 |
| Ni-NTA Superflow resin | Qiagen | Cat# 30430 |
| IPTG | Sigma-Aldrich | Cat# I5502 |
| Lysozyme from chicken egg white | Sigma-Aldrich | Cat# L6876 |
| Ubiquitin Activating Enzyme (E1) | R&D Systems | Cat# E-305–025 |
| UBE2D2 (from E2 Screening Kit) | UBPBio | Cat# J1100 |
| UBE2E1 (from E2 Screening Kit) | UBPBio | Cat# J1100 |
| Recombinant Human Ubiquitin Protein, CF | R&D Systems | Cat# U-100H |
| ESF 921 Insect Ceii Cuiture Medium | Expression Systems | Cat# 96–001-01 |
| Amyiose resin | NEB | Cat# E8021L |
| Leupeptin | RPI | Cat# L22035–0.050 |
| Pepstatin A | AdipoGen | Cat# AGCP37001M100 |
| PMSF | RPI | Cat# P20270–25.0 |
| Benzamidine | RPI | Cat# B12000–100.0 |
| Adenosine 5’-diphosphate sodium sait | Sigma-Aidrich | Cat# A2754 |
| Beryiiium suifate tetrahydrate | Sigma-Aidrich | Cat# 202789 |
| Sodium fiuoride | Sigma-Aidrich | Cat# 201154 |
| Giutaraidehyde | EMS | Cat# 16200 |
| Criticai commerciai assays | ||
| TnT SP6 High-Yield Wheat Germ Protein Expression System | Promega | Cat# L3260 |
| NEDD8 Conjugation Initiation Kit | R&D Systems | Cat# K-800 |
| Deposited data | ||
| Pol II TC-NER complex | This study | PDB: 9BZ0 |
| Map i (Poi II TC-NER complex) | This study | EMDB: EMD-45050 |
| Map ii (TC-NER factors) | This study | EMDB: EMD-47263 |
| Map iii (CSB) | This study | EMDB: EMD-47262 |
| Map iv (STK19) | This study | EMDB: EMD-47261 |
| Map v (DDB1-DDA1) | This study | EMDB: EMD-47266 |
| Map vi (UVSSA-DDB1-CSA) | This study | EMDB: EMD-47267 |
| Map vii (RPB4/7) | This study | EMDB: EMD-47271 |
| Map viii (DDA1) | This study | EMDB: EMD-47272 |
| Map ix (Composite map) | This study | EMDB: EMD-47273 |
| Experimental models: Cell lines | ||
| Sf21 cells | Expression Systems | Cat# 94–003S |
| Sf9 cells | Expression Systems | Cat# 94–001S |
| Tni (Hi5) cells | Expression Systems | Cat# 94–002S |
| Experimental models: Organisms/strains | ||
| Xenopus laevis (females) | Nasco | Cat# LM0053MX |
| Xenopus laevis (males) | Nasco | Cat# LM00715MX |
| Oligonucleotides | ||
| See Table S2 for oligonucleotides and gene blocks | - | N/A |
| Recombinant DNA | ||
| pTM07_5xUAS_AdMLΔ53_BbsI (referred to as pAdMLΔ53) | This study | N/A |
| pTM171_5xUAS_SCP2*_Ub_BbsI (referred to as pSCP2*) | This study | N/A |
| pTM180_5xUAS_SCP2*_Ub_BbsI_poiyA (referred to as pCtri-322) | This study | N/A |
| pTM181_5xUAS_SCP2*_BbsI_poiyA (referred to as pCtri-122) | This study | N/A |
| pTM210_5xUAS_SCP2*_Inverted-BbsI_poiyA (referred to as pCtri-NTS) | This study | N/A |
| pCMV-GFP (referred to as pCMV) | Matsuda & Cepko50 | Addgene Cat# 11153 |
| pActin | Barrows et al.34 | N/A |
| pOPINK | Gift from Ray Owens | Addgene Cat# 41143 |
| pOPINB | Gift from Ray Owens | Addgene Cat# 41142 |
| pRJR1_GAL4-VP64 | Stephen Buratowski | N/A |
| pET_hsTBP | Stephen Buratowski | N/A |
| pTM142_pAB1_FLAG-hsCSBWT | van der Weegen et al.15 | N/A |
| pTM143_pAB1_FLAG-hsCSBΔCIM | van der Weegen et al.15 | N/A |
| pTM449 pAB1_His6-TEV-Avi-xlDDB1_xlCSAWT | This study | N/A |
| pTM451 pAB1 His6-TEV-Avi-xlDDB1_xlCSAY–A | This study | N/A |
| pTM450 pAB1 His6-TEV-Avi-xlDDB1_xlCSAWS-AA | This study | N/A |
| pTM67_pAB1_FLAG-xlCUL4A_xlRBX1 | van der Weegen et al.15 | N/A |
| pTM185_pOPINK_xlELOF1WT | van der Weegen et al.17 | N/A |
| pTM194_pOPINK_xlELOF1SD-KK | This study | N/A |
| pTM288_pOPINK_xlELOF1NHE-AAA | This study | N/A |
| pTM81_pAB1_FLAG-xlUVSSAWT | This study | N/A |
| pTM186_pOPINK_xlSTK19WT | This study | N/A |
| pTM366_pOPINB_xlSTK19WT | This study | N/A |
| pTM376_pOPINB_xlSTK19ΔN | This study | N/A |
| pTM367_pOPINB_xlSTK194A | This study | N/A |
| pTM378_pOPINB_xlSTK19ΔN 4A | This study | N/A |
| pTM377_pOPINB_xlSTK19DR–RD | This study | N/A |
| pTM373_pOPINB_xlSTK19RY–GG | This study | N/A |
| pTM374_pOPINB_xlSTK19D–R | This study | N/A |
| pTM384_pOPINB_xlSTK19AS–EY | This study | N/A |
| pTM506_pOPINB_xlSTK19RYDAS–GGREY | This study | N/A |
| pTM408_pOPINB_xlSTK19R206A | This study | N/A |
| pTM409_pOPINB_xlSTK19R207A | This study | N/A |
| pTM399_pOPINB_xlSTK19RR–AA | This study | N/A |
| pTM380_pOPINB_xlSTK19RR–EE | This study | N/A |
| pTM365_pOPINB_hsSTK19WT | This study | N/A |
| pF3A | Promega | Cat# L5671 |
| pTM240_pF3A_xlUVSSA | This study | N/A |
| pTM242_pF3A_xlUVSSAΔCIR | This study | N/A |
| pTM248_pF3A_xlUVSSAΔTIR | This study | N/A |
| pTM252_pF3A_xlUVSSAFV–AA | This study | N/A |
| pTM266_pF3A_xlUVSSAE10A | This study | N/A |
| 438-A | Gradia et al.51 | Addgene Cat# 55218 |
| 438-B | Gradia et al.51 | Addgene Cat# 55219 |
| 438-C | Gradia et al.51 | Addgene Cat# 55220 |
| 1-B | Gradia et al.51 | Addgene Cat# 29653 |
| 1-C | Gradia et al.51 | Addgene Cat# 29654 |
| 438-C_NHis6-TEV-CSBV1097M, G1213R, R1413Q | This study | N/A |
| 438-B_NHis6-TEV-DDB1+ERCC8 | This study | N/A |
| 438-B_NHis6-TEV-UVSSA | This study | N/A |
| 1-B NHis6-TEV-ELOF1 | This study | N/A |
| 1-C_NHis6-MBP-N10-TEV-DDA1 | This study | N/A |
| Software and algorithms | ||
| ImageJ2 - Fiji (version 2.14) | Schindelin et ai.52 | https://imagej.net/software/fiji/ |
| GraphPad Prism (version 10.2.2) | GraphPad Software Inc. | https://www.graphpad.com/ |
| Octet Anaiysis Studio (version 13.0) | Sartorius | N/A |
| cryoSPARC (version 4.4.1) | Punjani et ai.53 | https://www.cryosparc.com |
| PHENIX (version 1.20.1) | Liebschner et ai.54 | https://phenix-oniine.org/ |
| ISOLDE (version 1.7.1) | Croii55 | https://tristanic.github.io/isolde/ |
| UCSF ChimeraX (versions 1.6–1.7) | Goddard et ai.56 | https://www.cgi.ucsf.edu/chimerax/ |
| Adobe Iiiustrator (version 28.7.1) | Adobe | https://www.adobe.com/products/iiiustrator.html |
| Other | ||
| HiLoad 16/600 Superdex 75 pg column | Cytiva | Cat# 28989333 |
| HiLoad 16/600 Superdex 200 pg column | Cytiva | Cat# 28989335 |
| Superdex 200 Increase 10/300 GL column | Cytiva | Cat# 28990944 |
| Superose 6 Increase 10/300 GL column | Cytiva | Cat# 29091596 |
| Superose 6 Increase 3.2/300 | Cytiva | Cat# 29091598 |
| Mono Q 5/50 GL column | Cytiva | Cat# 17516601 |
| GSTrap HP column | Cytiva | Cat# 17528202 |
| HisTrap HP column | Cytiva | Cat# 17524802 |
| HiTrap Q HP column | Cytiva | Cat# 17115301 |
| HiTrap Heparin HP column | Cytiva | Cat# 17040701 |
| HiTrap SP HP column | Cytiva | Cat# 17115101 |
| XK column 16/20 column | Cytiva | Cat# 28988937 |
| Typhoon FLA 7000 phosphorimager | GE Heaithcare | N/A |
| Typhoon 5 | GE Heaithcare | N/A |
| Amersham Imager 600 | GE Heaithcare | N/A |
| 4–15% Mini-PROTEAN TGX Precast Protein Gels | Bio-Rad | Cat# 4561086 |
| 4–15% Criterion TGX Precast Midi Protein Gels | Bio-Rad | Cat# 5671085 |
| 7.5% Mini-PROTEAN TGX Precast Protein Gels | Bio-Rad | Cat# 4561026 |
| NuPAGE 4–12% Bis-Tris protein gels | Invitrogen | Cat# NP0321BOX |
| PVDF membranes | VWR | Cat# PI88518 |
| Octet RED384 system | Sartorius | N/A |
| Octet Streptavidin (SA) Biosensor | Sartorius | Cat# 18–5019 |
| Octet 384 Well Tilted Bottom Plates | Sartorius | Cat# 18–5080 |
| UltrAuFoil R 2/2 (200 Mesh) | Quantifoii | Cat# N1-A16nAu20–01 |
| Vitrobot Mark IV | FEI/Thermo Fisher Scientific | N/A |
| Titan Krios + Falcon 4i + Selectris Energy Filter | FEI/Thermo Fisher Scientific | N/A |