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Structural basis for the initiation of eukaryotic transcription-coupled DNA repair

Abstract

Eukaryotic transcription-coupled repair (TCR) is an important and well-conserved sub-pathway of nucleotide excision repair that preferentially removes DNA lesions from the template strand that block translocation of RNA polymerase II (Pol II)1,2. Cockayne syndrome group B (CSB, also known as ERCC6) protein in humans (or its yeast orthologues, Rad26 in Saccharomyces cerevisiae and Rhp26 in Schizosaccharomyces pombe) is among the first proteins to be recruited to the lesion-arrested Pol II during the initiation of eukaryotic TCR1,3,4,5,6,7,8,9,10. Mutations in CSB are associated with the autosomal-recessive neurological disorder Cockayne syndrome, which is characterized by progeriod features, growth failure and photosensitivity1. The molecular mechanism of eukaryotic TCR initiation remains unclear, with several long-standing unanswered questions. How cells distinguish DNA lesion-arrested Pol II from other forms of arrested Pol II, the role of CSB in TCR initiation, and how CSB interacts with the arrested Pol II complex are all unknown. The lack of structures of CSB or the Pol II–CSB complex has hindered our ability to address these questions. Here we report the structure of the S. cerevisiae Pol II–Rad26 complex solved by cryo-electron microscopy. The structure reveals that Rad26 binds to the DNA upstream of Pol II, where it markedly alters its path. Our structural and functional data suggest that the conserved Swi2/Snf2-family core ATPase domain promotes the forward movement of Pol II, and elucidate key roles for Rad26 in both TCR and transcription elongation.

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Figure 1: Rad26 helps Pol II to discriminate among different transcription obstacles.
Figure 2: Cryo-electron microscopy structure of the Pol II EC bound to Rad26.
Figure 3: Rad26 binds to the upstream DNA and bubble fork of Pol II EC and bends the upstream DNA.
Figure 4: Rad26 translocates along the template strand towards Pol II.
Figure 5: Rad26 resolves Pol II backtracking in an ATP-dependent manner.

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References

  1. Hanawalt, P. C. & Spivak, G. Transcription-coupled DNA repair: two decades of progress and surprises. Nat. Rev. Mol. Cell Biol. 9, 958–970 (2008)

    Article  CAS  PubMed  Google Scholar 

  2. Svejstrup, J. Q. Contending with transcriptional arrest during RNAPII transcript elongation. Trends Biochem. Sci. 32, 165–171 (2007)

    Article  CAS  PubMed  Google Scholar 

  3. Troelstra, C. et al. ERCC6, a member of a subfamily of putative helicases, is involved in Cockayne’s syndrome and preferential repair of active genes. Cell 71, 939–953 (1992)

    Article  CAS  PubMed  Google Scholar 

  4. van Gool, A. J. et al. RAD26, the functional S. cerevisiae homolog of the Cockayne syndrome B gene ERCC6. EMBO J. 13, 5361–5369 (1994)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. van Gool, A. J. et al. The Cockayne syndrome B protein, involved in transcription-coupled DNA repair, resides in an RNA polymerase II-containing complex. EMBO J. 16, 5955–5965 (1997)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Tantin, D., Kansal, A. & Carey, M. Recruitment of the putative transcription-repair coupling factor CSB/ERCC6 to RNA polymerase II elongation complexes. Mol. Cell. Biol. 17, 6803–6814 (1997)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Selby, C. P. & Sancar, A. Human transcription-repair coupling factor CSB/ERCC6 is a DNA-stimulated ATPase but is not a helicase and does not disrupt the ternary transcription complex of stalled RNA polymerase II. J. Biol. Chem. 272, 1885–1890 (1997)

    Article  CAS  PubMed  Google Scholar 

  8. Sarker, A. H. et al. Recognition of RNA polymerase II and transcription bubbles by XPG, CSB, and TFIIH: insights for transcription-coupled repair and Cockayne Syndrome. Mol. Cell 20, 187–198 (2005)

    Article  CAS  PubMed  Google Scholar 

  9. Lainé, J. P. & Egly, J. M. Initiation of DNA repair mediated by a stalled RNA polymerase IIO. EMBO J. 25, 387–397 (2006)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Fousteri, M., Vermeulen, W., van Zeeland, A. A. & Mullenders, L. H. Cockayne syndrome A and B proteins differentially regulate recruitment of chromatin remodeling and repair factors to stalled RNA polymerase II in vivo. Mol. Cell 23, 471–482 (2006)

    Article  CAS  PubMed  Google Scholar 

  11. Lindsey-Boltz, L. A. & Sancar, A. RNA polymerase: the most specific damage recognition protein in cellular responses to DNA damage? Proc. Natl Acad. Sci. USA 104, 13213–13214 (2007)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  12. Saxowsky, T. T. & Doetsch, P. W. RNA polymerase encounters with DNA damage: transcription-coupled repair or transcriptional mutagenesis? Chem. Rev. 106, 474–488 (2006)

    Article  CAS  PubMed  Google Scholar 

  13. Belotserkovskii, B. P., Mirkin, S. M. & Hanawalt, P. C. DNA sequences that interfere with transcription: implications for genome function and stability. Chem. Rev. 113, 8620–8637 (2013)

    Article  CAS  PubMed  Google Scholar 

  14. Xu, L. et al. RNA polymerase II senses obstruction in the DNA minor groove via a conserved sensor motif. Proc. Natl Acad. Sci. USA 113, 12426–12431 (2016)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Sigurdsson, S., Dirac-Svejstrup, A. B. & Svejstrup, J. Q. Evidence that transcript cleavage is essential for RNA polymerase II transcription and cell viability. Mol. Cell 38, 202–210 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Selby, C. P. & Sancar, A. Cockayne syndrome group B protein enhances elongation by RNA polymerase II. Proc. Natl Acad. Sci. USA 94, 11205–11209 (1997)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  17. Brueckner, F., Hennecke, U., Carell, T. & Cramer, P. CPD damage recognition by transcribing RNA polymerase II. Science 315, 859–862 (2007)

    Article  ADS  CAS  PubMed  Google Scholar 

  18. Hantsche, M. & Cramer, P. Conserved RNA polymerase II initiation complex structure. Curr. Opin. Struct. Biol. 47, 17–22 (2017)

    Article  CAS  PubMed  Google Scholar 

  19. Xu, L. et al. RNA polymerase II transcriptional fidelity control and its functional interplay with DNA modifications. Crit. Rev. Biochem. Mol. Biol. 50, 503–519 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Martinez-Rucobo, F. W. & Cramer, P. Structural basis of transcription elongation. Biochim. Biophys. Acta 1829, 9–19 (2013)

    Article  CAS  PubMed  Google Scholar 

  21. Thomä, N. H. et al. Structure of the SWI2/SNF2 chromatin-remodeling domain of eukaryotic Rad54. Nat. Struct. Mol. Biol. 12, 350–356 (2005)

    Article  PubMed  CAS  Google Scholar 

  22. Liu, X., Li, M., Xia, X., Li, X. & Chen, Z. Mechanism of chromatin remodelling revealed by the Snf2–nucleosome structure. Nature 544, 440–445 (2017)

    Article  ADS  CAS  PubMed  Google Scholar 

  23. Ehara, H. et al. Structure of the complete elongation complex of RNA polymerase II with basal factors. Science 357, 921–924 (2017)

    Article  ADS  CAS  PubMed  Google Scholar 

  24. Li, W., Giles, C. & Li, S. Insights into how Spt5 functions in transcription elongation and repressing transcription coupled DNA repair. Nucleic Acids Res. 42, 7069–7083 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Jansen, L. E. et al. Spt4 modulates Rad26 requirement in transcription-coupled nucleotide excision repair. EMBO J. 19, 6498–6507 (2000)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Charlet-Berguerand, N. et al. RNA polymerase II bypass of oxidative DNA damage is regulated by transcription elongation factors. EMBO J. 25, 5481–5491 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Muftuoglu, M. et al. Cockayne syndrome group B protein has novel strand annealing and exchange activities. Nucleic Acids Res. 34, 295–304 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Bernecky, C., Herzog, F., Baumeister, W., Plitzko, J. M. & Cramer, P. Structure of transcribing mammalian RNA polymerase II. Nature 529, 551–554 (2016)

    Article  ADS  CAS  PubMed  Google Scholar 

  29. Deaconescu, A. M. et al. Structural basis for bacterial transcription-coupled DNA repair. Cell 124, 507–520 (2006)

    Article  CAS  PubMed  Google Scholar 

  30. Li, C. L. et al. Tripartite DNA lesion recognition and verification by XPC, TFIIH, and XPAin nucleotide excision repair. Mol. Cell 59, 1025–1034 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Wang, D., Bushnell, D. A., Westover, K. D., Kaplan, C. D. & Kornberg, R. D. Structural basis of transcription: role of the trigger loop in substrate specificity and catalysis. Cell 127, 941–954 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Wang, L. et al. Molecular basis for 5-carboxycytosine recognition by RNA polymerase II elongation complex. Nature 523, 621–625 (2015)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  33. Kireeva, M. L., Komissarova, N., Waugh, D. S. & Kashlev, M. The 8-nucleotide-long RNA:DNA hybrid is a primary stability determinant of the RNA polymerase II elongation complex. J. Biol. Chem. 275, 6530–6536 (2000)

    Article  CAS  PubMed  Google Scholar 

  34. Suloway, C. et al. Automated molecular microscopy: the new Leginon system. J. Struct. Biol. 151, 41–60 (2005)

    CAS  PubMed  Google Scholar 

  35. Plaschka, C. et al. Transcription initiation complex structures elucidate DNA opening. Nature 533, 353–358 (2016)

    Article  ADS  CAS  PubMed  Google Scholar 

  36. Plaschka, C. et al. Architecture of the RNA polymerase II–Mediator core initiation complex. Nature 518, 376–380 (2015)

    Article  ADS  CAS  PubMed  Google Scholar 

  37. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Mindell, J. A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003)

    Article  PubMed  Google Scholar 

  39. Voss, N. R., Yoshioka, C. K., Radermacher, M., Potter, C. S. & Carragher, B. DoG Picker and TiltPicker: software tools to facilitate particle selection in single particle electron microscopy. J. Struct. Biol. 166, 205–213 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Bharat, T. A. & Scheres, S. H. Resolving macromolecular structures from electron cryo-tomography data using subtomogram averaging in RELION. Nat. Protocols 11, 2054–2065 (2016)

    Article  CAS  PubMed  Google Scholar 

  42. Roseman, A. M. FindEM—a fast, efficient program for automatic selection of particles from electron micrographs. J. Struct. Biol. 145, 91–99 (2004)

    Article  CAS  PubMed  Google Scholar 

  43. Lander, G. C. et al. Appion: an integrated, database-driven pipeline to facilitate EM image processing. J. Struct. Biol. 166, 95–102 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Kimanius, D., Forsberg, B. O., Scheres, S. H. & Lindahl, E. Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. eLife 5, e18722 (2016)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Cianfrocco, M. A. & Leschziner, A. E. Low cost, high performance processing of single particle cryo-electron microscopy data in the cloud. eLife 4, e06664 (2015)

    Article  PubMed Central  Google Scholar 

  46. Kettenberger, H., Armache, K.-J. & Cramer, P. Complete RNA polymerase II elongation complex structure and its interactions with NTP and TFIIS. Mol. Cell 16, 955–965 (2004)

    Article  CAS  PubMed  Google Scholar 

  47. Scheres, S. H. & Chen, S. Prevention of overfitting in cryo-EM structure determination. Nat. Methods 9, 853–854 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Henderson, R. et al. Outcome of the first electron microscopy validation task force meeting. Structure 20, 205–214 (2012)

    Article  CAS  PubMed  Google Scholar 

  49. Chen, S. et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 24–35 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Bai, X. C., Rajendra, E., Yang, G., Shi, Y. & Scheres, S. H. Sampling the conformational space of the catalytic subunit of human γ-secretase. eLife 4, e11182 (2015)

    Article  PubMed  PubMed Central  Google Scholar 

  51. Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015)

    Article  PubMed  PubMed Central  Google Scholar 

  52. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017)

    Article  CAS  PubMed  Google Scholar 

  53. Wang, R. Y. et al. Automated structure refinement of macromolecular assemblies from cryo-EM maps using Rosetta. eLife 5, e17219 (2016)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. Song, Y. et al. High-resolution comparative modeling with RosettaCM. Structure 21, 1735–1742 (2013)

    Article  CAS  PubMed  Google Scholar 

  55. DiMaio, F. et al. Atomic-accuracy models from 4.5-Å cryo-electron microscopy data with density-guided iterative local refinement. Nat. Methods 12, 361–365 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Söding, J., Biegert, A. & Lupas, A. N. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res. 33, W244–W248 (2005)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  57. Barnes, C. O. et al. Crystal structure of a transcribing RNA polymerase II complex reveals a complete transcription bubble. Mol. Cell 59, 258–269 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

    Google Scholar 

  59. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

    Article  CAS  PubMed  Google Scholar 

  60. Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

    CAS  PubMed  Google Scholar 

  61. Pintilie, G. D., Zhang, J., Goddard, T. D., Chiu, W. & Gossard, D. C. Quantitative analysis of cryo-EM density map segmentation by watershed and scale-space filtering, and fitting of structures by alignment to regions. J. Struct. Biol. 170, 427–438 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank the Wang and Leschziner laboratories for discussions. D.W., A.E.L. and P.B.D. were supported by National Institutes of Health (NIH) grants GM102362, GM102362-S1 (D.W.), GM092895 (A.E.L.), and GM27681 (P.B.D.). M.A.C. acknowledges support from the Damon Runyon Cancer Research Foundation. We thank the UCSD cryo-EM Facility, where all data was collected. We used the Extreme Science and Engineering Discovery Environment (XSEDE) for computing allocations (MCB160121 to D.W.), supported by NSF grant ACI-1548562.

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Authors

Contributions

J.X. prepared the proteins with help from W.W. and J.C. and performed the biochemical analyses. A.H. and P.D.B. provided the Py-Im chemical agent. I. L. collected the EM data with help from A.W. I.L. performed data processing and refinement with help from M.A.C. I.L. and F.D. generated the atomic models with homology models generated by J.X., W.W. and D.W. D.W. and A.E.L. wrote the manuscript with help from all laboratory members. D.W. and A.E.L. directed and supervised the research.

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Correspondence to Andres E. Leschziner or Dong Wang.

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Reviewer Information Nature thanks A. Conconi and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Sequence alignment of the ATPase core domains of CSB family members.

Protein sequences from the CSB ATPase core region from S. cerevisiae, S. pombe, Arabidopsis thaliana, Danio rerio, Mus musculus and Homo sapiens were aligned using Clustal Omega. Residues are numbered based on the sequence of the S. cerevisiae CSB orthologue Rad26. Conserved residues are highlighted in red and helicase-specific motifs are boxed in black and labelled with roman numerals. The flexible disordered loop regions that were not built into the cryo-EM density are indicated, as are the Swi2/Snf2-specific domains HD1 and HD2.

Extended Data Figure 2 Cryo-EM reconstructions of the Pol II–Rad26 and Pol II EC complexes.

a, Representative micrograph of Pol II–Rad26 complexes. Scale bar, 100 nm. b, Power spectrum of the micrograph in a showing Thon rings out to 3.4 Å. c, Representative 2D class averages of the Pol II–Rad26 complex. d, Schematic representation of the strategy used to sort out the datasets into Pol II EC and Pol II–Rad26 complex structures. Unless otherwise noted, 3D classification was performed without image alignment. Coloured, segmented maps indicate classes in which particles were used for further processing. The colour scheme used in the segmented maps is as follows: Pol II (grey), Rad26 (orange), transcription scaffold (green). Black lines follow the classification scheme used to extract homogeneous Pol II–Rad26 particles; blue lines follow the classification scheme used to extract homogeneous Pol II EC particles. The refined maps for the higher-resolution Pol II–Rad26 complex (with fragmented Rad26 density), final Pol II–Rad26 complex and Pol II EC are highlighted with green, black and blue boxes, respectively. The indicated resolution corresponds to the 0.143 Fourier shell correlation (FSC) based on gold-standard FSC curves. The number of particles contributing to each selected structure is indicated. The percentages shown are relative to the total number of particles selected after 2D classification. eg, Front and back views of locally filtered maps coloured by local resolution. h, Euler angle distribution of particle images for the maps shown in eg. i, FSC plots for the higher-resolution Pol II–Rad26 complex (with fragmented Rad26 density), final Pol II–Rad26 complex and Pol II EC maps with the resolution at 0.143 FSC indicated. j, Representative near-atomic resolution regions in Pol II from the locally filtered higher-resolution (4.5 Å) Pol II–Rad26 map. The density is shown in transparent grey with the atomic model for Pol II–Rad26 complex fitted in the map. The β-sheet corresponds to residues 346–356, 440–446 and 486–493 in Rpb1, and 1104–1107 in Rpb2. The portion of the bridge helix shown here corresponds to residues 810–829 in Rpb1.

Extended Data Figure 3 Validation of Rosetta models for the Pol II–Rad26 complex and Pol II EC.

a, Table summarizing the main statistics from data collection, refinement and model validation. b, c, The r.m.s.d. values of the protein backbones among the top five conformations (based on Rosetta energy) of the Pol II–Rad26 complex (b) and Pol II EC (c) generated by RosettaCM. In both cases the best Rosetta energy model is shown as a worm model, with thickness and colour representing the backbone r.m.s.d. value. The transcription scaffolds were not included in the r.m.s.d. calculation and were omitted for clarity. d, Backbone r.m.s.d. values between the atomic models of Pol II–Rad26 complex and Pol II EC shown on the atomic model of Pol II–Rad26 complex using the same representation as in b and c. The models were globally aligned to each other in Chimera (UCSF) and only those parts of the model for which r.m.s.d. calculation could be performed are shown. e, f, FSC curves between the atomic model and cryo-EM maps for the Pol II–Rad26 complex (e) and Pol II EC (f). In e, FSCwork and FSCfree values were calculated using half maps from the higher-resolution Pol II–Rad26 complex structure. The 0.5 FSC line is shown. g, h, Three different views of the Pol II–Rad26 map with models docked in (g), and close-up views of the Pol II–Rad26 interface (h).

Extended Data Figure 4 Cryo-EM reconstruction of a Pol II EC containing a CPD lesion.

a, Representative micrograph of Pol II EC (CPD). b, Power spectrum of the micrograph in a. c, Representative 2D class averages of the Pol II EC (CPD) complex. d, FSC plot for the final Pol II EC (CPD) map with the resolution at 0.5 FSC indicated. e, Euler angle distribution of particle images. f, Table summarizing data collection statistics. gk, Strategy for generating difference map between Pol II–Rad26 and Pol II EC (CPD). We took the model for the Pol II–Rad26 complex (g), removed Rad26 (h), and converted the resulting model into a cryo-EM-like density (i). From this, we subtracted the Pol II EC (CPD) map (j) to obtain the difference map (k). l, Two views of the Pol II EC (CPD) map. m, Model of the Pol II EC complex after removal of Rad26 (h) docked into the Pol II EC (CPD) map. n, Same as in m, with the difference map superimposed.

Extended Data Figure 5 Alignment of the HD2-1 region of CSB and non-CSB members of the Swi2/Snf2 superfamily of ATPases.

The HD2-1 region corresponds to the wedge motif in the Pol II–Rad26 structure (see Fig. 3e). See Extended Data Fig. 1 for the location of the HD2-1 region within the full ATPase domain. Residues are coloured (according to physicochemical properties) when conserved in at least half of the sequences.

Extended Data Figure 6 The strength of base pairing at the upstream fork of the transcription bubble, not CPD lesions at downstream fork, affects the interaction of Rad26 with Pol II EC.

a, The sequence of the scaffold used in this study. The nucleotides labelled as XXX and YYY were varied in these experiments to control the strength of the base pairing at the upstream fork of the transcription bubble. b, Electrophoretic mobility shift assay (EMSA) between Rad26 and Pol II EC with an AT-rich sequence at the upstream fork of the DNA bubble. c, EMSA between Rad26 and Pol II EC with a GC-rich sequence at the upstream fork of the DNA bubble. d, Quantification of the assays shown in b, c. Data are mean and s.d. (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, two-tailed Student’s t-test. Precise P values shown in Extended Data Table 1. e, Modelled structure of Pol II in complex with the mini-scaffold. Rad26, from the Pol II–Rad26 complex structure, was included as a semi-transparent ribbon diagram to indicate the lack of interaction between it and the mini-scaffold. Mini-scaffolds that eliminate the upstream DNA to which Rad26 binds were used to form elongation complexes (mini-ECs) with Pol II, and the interaction between these mini-ECs and Rad26 was tested using EMSA. f, DNA/RNA scaffolds used in this experiment. To rule out the possibility that Rad26 may bind to dsDNA in a non-specific manner, a scaffold with only RNA and template strand (scaffold 2) was also tested. g, h, EMSA with scaffold 1 (g) and scaffold 2 (h) showing formation of a Pol II mini-EC–Rad26 complex. The experiment was repeated independently twice with similar results. i, Scaffolds with or without a CPD lesion (see Methods for details) were used to form elongation complexes with Pol II, and the interaction between them and Rad26 was tested using EMSA. j, Quantification of data in i. Data are mean and s.d. (n = 3). All biochemical experiments were repeated independently 3 times with similar results, except 2 times for g and h. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 7 Overlap between the binding sites of Rad26 and Spt4–Spt5 on Pol II.

a, c, Structure of the Pol II–Rad26 complex, with Rad26 and the DNA/RNA scaffold shown in surface representation. b, d, Structure of Pol II EC bound to Spt4–Spt5 and TFIIS (PDB accession 5XON), with Spt4 and Spt5 shown in surface representation. The different domains of Spt5 are indicated. e, Rad26 and the DNA/RNA scaffold from a are superimposed on Spt4–Spt5 from b. f, Rotated view of e. g, Rad26 and the DNA/RNA scaffold from c are superimposed on Spt4–Spt5 from d. h, Rotated view of g. The bicolour arrows indicate clashes between Rad26 or the DNA/RNA scaffold and Spt4–Spt5.

Extended Data Figure 8 Alignment between Snf2 and Rad26.

a, This panel is identical to Fig. 4b and is included here as a reference. b, Superposition between Rad26, bound to the transcription scaffold, and Snf2 from the cryo-EM structure of the Snf2–nucleosome complex (PDB accession 5X0Y), with the nucleosome included in the image. This is the same alignment shown in Fig. 4a–c and in a, and was driven exclusively by Snf2 and Rad26. This view is rotated by 180° about the vertical axis relative to a. The dashed box marks the portion of the structure equivalent to that shown in a. The back gyre of the nucleosome was faded out for clarity. c, Same view as in b, with Snf2 and Rad26 removed to illustrate the superposition of the Rad26-bound portion of the transcription scaffold and the nucleosomal DNA. dg, Alignment of Rad26 and Snf2. The superimposed structures are shown in two orientations (d, f), with d corresponding to the direction indicated by the symbol in a. A worm model is used to represent the similarity between the two structures (e, g), with thickness and colour indicating the backbone r.m.s.d. value. The thin wire corresponds to regions in the Rad26 model that are not present in Snf2.

Extended Data Figure 9 Unified model for three-step DNA lesion recognition and verification for both TCR and GG-NER.

Check step 1: for GG-NER, XPC or HR23B detects base-pair disruption and helix distortion and binds to the DNA strand opposite that carrying the lesion. This constitutes the initial lesion recognition. For TCR, CSB is recruited to a stalled Pol II to discriminate genuine DNA lesion-induced transcription arrest from other forms of transcriptional arrest. At this step, CSB acts in conjunction with Pol II to mediate the initial recognition of DNA lesions that block transcription translocation. Check step 2: core TFIIH is recruited to verify the DNA lesion further. In GG-NER, the XPD and XPB helicases in core TFIIH translocate the complex towards the lesion. This is the result of XPD tracking along the damage-containing strand in a 5′-to-3′ direction, and XPB tracking along the opposite (non-damaged) strand in a 3′-to-5′ direction. In TCR, TFIIH is loaded downstream of the arrested Pol II–CSB complex, with XPD and XPB tracking the template and non-template strands, respectively. The XPD and XPB helicases in core TFIIH translocate towards the lesion, as is the case for GG-NER. As a result, Pol II–CSB is pushed upstream by TFIIH to expose the DNA lesion. Check step 3: XPA is recruited for a final validation of the TFIIH-recognized lesion and to ensure that only genuine NER lesions are subjected to dual incision by endonucleases ERCC1–XPF and XPG and downstream repair synthesis.

Extended Data Table 1 Specific P values

Supplementary information

Supplementary Information

This file contains Supplementary Figure 1, the gel source data and Supplementary Tables 1-2. (PDF 1773 kb)

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Xu, J., Lahiri, I., Wang, W. et al. Structural basis for the initiation of eukaryotic transcription-coupled DNA repair. Nature 551, 653–657 (2017). https://doi.org/10.1038/nature24658

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