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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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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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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The authors declare no competing financial interests.

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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

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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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