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Crucial role and mechanism of transcription-coupled DNA repair in bacteria

Nature volume 604, pages 152–159 (2022)Cite this article

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Abstract

Transcription-coupled DNA repair (TCR) is presumed to be a minor sub-pathway of nucleotide excision repair (NER) in bacteria. Global genomic repair is thought to perform the bulk of repair independently of transcription. TCR is also believed to be mediated exclusively by Mfd—a DNA translocase of a marginal NER phenotype1,2,3. Here we combined in cellulo cross-linking mass spectrometry with structural, biochemical and genetic approaches to map the interactions within the TCR complex (TCRC) and to determine the actual sequence of events that leads to NER in vivo. We show that RNA polymerase (RNAP) serves as the primary sensor of DNA damage and acts as a platform for the recruitment of NER enzymes. UvrA and UvrD associate with RNAP continuously, forming a surveillance pre-TCRC. In response to DNA damage, pre-TCRC recruits a second UvrD monomer to form a helicase-competent UvrD dimer that promotes backtracking of the TCRC. The weakening of UvrD–RNAP interactions renders cells sensitive to genotoxic stress. TCRC then recruits a second UvrA molecule and UvrB to initiate the repair process. Contrary to the conventional view, we show that TCR accounts for the vast majority of chromosomal repair events; that is, TCR thoroughly dominates over global genomic repair. We also show that TCR is largely independent of Mfd. We propose that Mfd has an indirect role in this process: it participates in removing obstructive RNAPs in front of TCRCs and also in recovering TCRCs from backtracking after repair has been completed.

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Fig. 1: Recruitment of NER enzymes and Mfd to RNAP in vivo.
Fig. 2: Structural organization of the pre-TCR and TCR complexes in cellulo.
Fig. 3: Structure and function of the UvrD CTD–RNAP β pincer complex.
Fig. 4: Global NER fully depends on ongoing transcription and UvrD, but not on Mfd.
Fig. 5: Local transcription enables NER (independently of Mfd).

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Data availability

Coordinates and structure factors of the X-ray crystallography structures determined in this study have been deposited in the PDB under accession numbers 7EGT and 7EGS, and are listed in Extended Data Table 1. PDB codes for the XLMS-driven docking models are available at http://nudlerlab.info/media/TCRC_computational_models.zip.

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Acknowledgements

We thank the staff at beamline BL17U1/BL18U1/BL19U1 of Shanghai Synchrotron Radiation Facility for assistance during data collection, and T. Artemev for his continued support. The work was supported by the National Key Research and Development Program of China (grant no. 2018YFA0903701), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB29020302), the Chinese Natural Science Foundation of China (31822001) and the Shanghai Science and Technology Innovation program (19JC1415900) (Y.Z.), and by NIH grant R01 GM126891, the Blavatnik Family Foundation and the Howard Hughes Medical Institute (E.N.).

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Author notes

  1. These authors contributed equally: Binod K. Bharati, Manjunath Gowder

Authors and Affiliations

  1. Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY, USA

    Binod K. Bharati, Manjunath Gowder, Khaled Alzoubi, Vladimir Svetlov, Venu Kamarthapu, Jacob W. Weaver, Vitaly Epshtein, Nikita Vasilyev & Evgeny Nudler

  2. Howard Hughes Medical Institute, New York University School of Medicine, New York, NY, USA

    Binod K. Bharati, Manjunath Gowder, Venu Kamarthapu & Evgeny Nudler

  3. Key Laboratory of Synthetic Biology, Chinese Academy of Sciences Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China

    Fangfang Zheng, Liqiang Shen & Yu Zhang

Authors
  1. Binod K. Bharati
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  2. Manjunath Gowder
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  3. Fangfang Zheng
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  4. Khaled Alzoubi
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  5. Vladimir Svetlov
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  6. Venu Kamarthapu
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  7. Jacob W. Weaver
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  8. Vitaly Epshtein
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  9. Nikita Vasilyev
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  10. Liqiang Shen
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  11. Yu Zhang
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  12. Evgeny Nudler
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Contributions

B.K.B. generated bacterial strains, performed the pull-down experiments described in Fig. 1 and performed CPD repair and other experiments described in Fig. 4 and Extended Data Figs. 1, 4–6. M.G. generated bacterial strains and performed DNA repair and other experiments described in Fig. 5 and Extended Data Figs. 6–8, 10. F.Z. and L.S. purified and crystallized the proteins, determined the X-ray crystal structures and performed other experiments described in Fig. 3a and Extended Data Fig. 3. K.A. performed XLMS-driven structural docking and modelling. V.S. performed XLMS and DLS experiments. V.K. and J.W.W. purified the proteins and reconstituted pre-TCRCs and TCRCs. V.K. and B.K.B. performed bacterial survival tests. V.E. performed in vitro transcription assays and UvrD biochemical analysis. N.V. performed mass-spectrometry analysis. Y.Z. supervised the structural work and assisted with data analysis. E.N. designed and supervised the project and wrote the manuscript with input from all authors. All authors discussed the results.

Corresponding authors

Correspondence to Yu Zhang or Evgeny Nudler.

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Extended data figures and tables

Extended Data Fig. 1 Quantification of UvrABD binding to RNAP in vivo.

Supplementary to Fig. 1. a, UV sensitivity of E. coli strains used in Fig. 1. Representative efficiencies of colony formation of parent wild-type (MG1655) and mutant E. coli cells exposed to the indicated UV doses. Overnight cultures were diluted 1:100 with fresh LB and grown to ~ 2x107. Cells were serially diluted, plated on LB agar, irradiated with UV, and incubated at 37 °C for 24 h. b, Representative western blots used to generate the plots of Fig. 1b–d. c, Quantitative mass-spectrometry analysis of RNAP-associated UvrABD, Mfd, and Rho in the exponentially grown cells prior to genotoxic stress. RNAP pulldown samples were prepared as in Fig. 1. Values are normalized to that of NusA-containing RNAPs, thus reflecting the RNAP molecules engaged in elongation in vivo.

Extended Data Fig. 2 XLMS-driven structural modelling of TCRC.

Supplementary to Fig. 2. a–d, Reconstitution of the TCRC without nucleic acids. a, Isolation of the RNAP–NusA–UvrABD complex by SEC. SDS-Coomassie gel represents the protein fraction eluted from the main peak (P). b, DLS analysis of the RNAP–NusA–UvrABD complex. The ‘P’ fraction from a was subjected to DLS. Raleigh sphere (R) estimate of the complex molecular weight (MW = 908 kDa), which deviates by only 1.7% from the theoretical MW of a uniform monodispersed complex containing 1 RNAP, 1 NusA, 2 UvrD, 2 UvrA, and 1 UvrB molecules. c, Network view of the highly confident non-redundant inter-protein cross-links (Supplementary Table 1). d, XLMS-based model of the reconstituted RNAP:NusA:UvrABD complex. The model was built based on the in vitro cross-links using PatchDock and the workflow described in Extended Data Fig. 11 and Methods. e–h, Mapping UvrD-EC interactions in vitro. e, Isolation of the EC:NusA:UvrD complex by SEC. SDS-Coomassie gel represents the protein fraction eluted from the main SEC peak (P). f, DLS analysis of the EC:NusA:UvrD complex. “P” fraction from (e) was subjected to DLS. Raleigh sphere (R) estimate of the complex molecular weight (MW = 620 kDa), which deviates by only 2% from the theoretical MW of a uniform monodispersed complex containing 1 RNAP, 1 NusA, and 2 UvrD molecules. g, Network view of the highly confident non-redundant inter-protein cross-links between RNAP subunits, NusA, and UvrD (Supplementary Table 1). h, XLMS-based model of the RNAP:NusA:UvrD complex (Extended Data Fig. 11 and Methods). The model shows the positioning of UvrD monomers relative to the transcription bubble. Blue star indicates the DNA-UvrD cross-link previously mapped in the EC18. CTD of UvrD2 is shown by green hexagon. RNA is not shown.

Extended Data Fig. 3 Structural interrogation of UvrD CTD interactions in TCRC.

Supplementary to Fig. 3a. a–g, Structural analysis of UvrD CTD–RNAP interactions. a, UvrD and RNAP β-subunit domains observed in crystals structure were coloured and labelled. b, The overall structure of the UvrD CTD–RNAP β2i4 (PDB: 7EGS, Extended Data Table 1) complex. The major interface is highlighted by a rectangle. The ‘N’ and ‘C’ termini of UvrD CTD are numbered. c, Detailed UvrD CTD–RNAP β2i4 interactions. Oxygen, nitrogen, and water atoms are coloured in red, blue, and orange, respectively. Blue dash, H-bond. d, Yeast two-hybrid assay results show that alanine substitution of interface residues on UvrD CTD or βi4 impairs the interaction of UvrD and RNAP β pincer. The potential interactions were selected on SD (-HALW) plates, and the growth on SD (-LW) plates was used as input control. e, Strep-tag pull down results show that alanine substitution of interface residues of UvrD CTD or βi4 impairs RNAP–UvrD interaction. f, Sequence alignments of UvrD CTD and RNAP β2i4 from 316 non-redundant proteobacteria that contain βi4 insertion on RNAP. Key interface residues were labelled with asterisks and numbered as in E. coli. g, Cys pair cross-linking results demonstrate direct proximity of UvrD CTD and RNAP βi4. Wild-type or mutated UvrD-RNAP complexes were incubated in oxidative (CuCl2) or reducing (DTT) condition and separated by SDS-PAGE.The asterisk marks two major impurity bands. h–m, Structural analysis of UvrD CTD–UvrB interactions. h, UvrD and UvrB domains (numbered as in E. coli). The domains observed in crystal structure are highlighted in colours. i, UvrD CTD interacts with UvrB-1a/1b/2domain (or UvrD NTD), consistent with a previous report35. E. coli UvrD and UvrB were fused to GAL4-AD and GAL4-DBD, respectively. The potential interactions were selected on SD (-HALW) plates, and the growth on SD (-LW) plates was used as input control. j, A 2.6-Å crystal structure of T. thermophilus UvrD CTD–UvrB NTD complex (PDB: 7EGT, Extended Data Table 1). UvrB-1a docks on a shallow groove of UvrD CTD. UvrD CTD, UvrB-1a, UvrB-1b, and UvrB-2 are coloured in purple, cyan, orange, and light green, respectively. k, Detailed UvrD CTD–UvrB-1a interactions. Residues H654, R656, K680, R681, S683 of UvrD CTD make a H-bond network with D27, E29, R30, Q383 of UvrB-1a (residues are numbered as in T. thermophilus; the corresponding residues in E. coli are indicated in parentheses). Y686 of UvrD CTD makes stacking interaction with R55 and Q383 of UvrB-1a. l, Structural superimposition of UvrB NTD/UvrD CTD complex (coloured as above) and UvrB/dsDNA complex (grey and red; PDB: 6O8F)102 shows that UvrD CTD binds the opposite surface of UvrB dsDNA-loading cleft, implicating UvrD doesn’t affect dsDNA loading Of UvrB. m, Structural comparison between UvrD CTD/UvrB NTD (left) and UvrD CTD/RNAP β2i4 (right, PDB: 7EGS) shows that UvrB and RNAP β2i4 binds at the same cleft of UvrD CTD, and thereby suggests that the interactions of UvrB and RNAP to UvrD are mutually exclusive.

Extended Data Fig. 4 Functional analysis of UvrD(ΔCTD) and RNAP(Δβi4).

Supplementary to Fig. 3. a, Deletions of the CTD of UvrD or βi4 of RNAP partially compromise UvrD-mediated backtracking. EC20 was formed by the wild-type RNAP or RNAP lacking βi4 (green Δ) (lanes 13 to 18) at the T7A1 DNA template and then chased in the presence of specified amounts of UvrD (red Δ). The pro-backtracking activity of UvrD was assessed as a ratio (%) between the amount of full length (run-off) product and total amounts of RNA products located below the run-off. Majority of these products are the result of UvrD-mediated backtracking and sensitive to transcript cleavage by GreB12,18. b, Deletion of the CTD does not compromise UvrD catalytic activity. The autoradiogram shows the thin layer chromatography (TLC) plate of UvrD-mediated ATP hydrolysis. The reaction was performed using polyC single stranded DNA template as described in Methods. The means ± SE from three experiments are plotted on the right. c, uvrDΔCTD and Δβi4 cells are equally more sensitive to genotoxic stress as compared to wild type. Representative efficiencies of colony formation of wild-type (MG1655) and mutant cells following treatment with the indicated dose of UV irradiation. Cells were grown to OD600 nm ~0.4 and serial 10-fold dilutions were spotted on LB agar plates followed by UV irradiation and incubation in the dark at 37 °C for 24 h.

Extended Data Fig. 5  NER strictly coupled to transcription and is mostly independent of Mfd.

Supplementary to Fig. 4. a, Wild-type (wt) and mutant cells were UV irradiated at 50 J/m2 and allowed to recover. At the indicated times, genomic DNA was isolated and either treated with T4endoV or mock treated for 30 min at 37 °C and then analysed on alkali-agarose gels. Rifampicin (Rif; 750 µg/ml), chloramphenicol (Cm; 200 µg/ml) and/or bicyclomycin (Bcm; 25 µg/ml) were added 30 min prior to UV irradiation (see Methods). Representative gels are shown for each analysed strain and condition. The percentage of repaired (lesion-free) DNA in T4endoV-treated samples is plotted for each time point relative to untreated samples (see Fig. 4). Data are the mean ± SEM from at least three independent experiments. b, Mfd overexpression interferes with global NER. The wild-type cells containing pMfd and the “empty” vector control (pCA24N) were induced with 0.1 mM IPTG followed by UV irradiation and recovery. The percentage of repaired (lesion-free) DNA in T4endoV-treated samples is plotted for each time point relative to untreated samples. Data are the mean ± SEM from at least three independent experiments.

Extended Data Fig. 6 High Rif abolishes NER.

Supplementary to Fig. 4. a, b, Effects of high (750 µg/ml) and low (50 µg/ml) Rif on E. coli transcription and NER. a, Inhibition of chromosomal lacZ transcription by Rif. Copies/μL cDNA of lacZ transcripts was determined using absolute quantification (see Methods). A standard curve was generated using lacZ PCR product (1016 to 1023). RT-qPCR was performed using cDNA isolated from bacterial cultures treated with indicated concentrations of Rif. Number of copies of lacZ transcripts was determined by interpolation. Values are the mean ± SD from 3 independent experiments. b, Inhibition of CPDs repair by high and low Rif. (Left panel) Representative slot blot probed by fluorescently labelled secondary antibody to reveal binding of primary monoclonal CPD-specific antibodies (see Methods). (Right panel) Quantitative analysis of slot blot images for the indicated recovery time points post-UV. Bars, standard errors of the means from 3 independent experiments. c, d, Rif does not compromise the level of NER enzymes during the time of the experiment. c, Representative western blots of intracellular UvrABCD proteins during the time of high Rif treatment (Fig. 4) and their quantification (d). Data are the mean ± SEM from at least three independent experiments.

Extended Data Fig. 7 Local transcription enables NER.

Supplementary to Fig. 5. a–d, Depriving the genomic loci of transcription abolishes their NER. a, Schematics of the mCherry insulators. A transcription unit containing mCherry (with or without lacZ promoter) flanked by the terminator cassettes was inserted at the tam and nupG loci. b–d The expression of mCherry from the insulators (b) and RNAP occupancy (c, d) after IPTG induction, as determined by RT-qPCR and ChIP–qPCR, respectively. Values are the mean ± SD from 3 independent experiments. e, f, CPD repair within the insulators. Most of NER strictly depends on promoter-initiated transcription. The levels of transcription and NER are stronger within the nupG insulator comparing to the tam insulator. Cells were induced with IPTG followed by UV irradiation (40 J/m2) and recovery in the dark for the indicated time intervals. CPD density was determined by SLR-qPCR as in Fig. 5a and used to calculate the percentage of repaired CPDs. Values are the mean ± SD from 3 independent experiments. g–j, UvrAB recruitment to the UV-damaged DNA strictly depends on local transcription in both tam and nupG loci. Occupancy of RNAP (c, d), UvrA (g, i) and UvrB (h, j) following UV irradiation was determined by ChIP–qPCR. Cells were induced with IPTG followed by UV irradiation (40 J/m2) and recovery for the indicated time intervals. Values are the mean ± SD from 3 independent experiments. Results are shown as a fold change in the occupancy of UvrAB within the insulator following UV irradiation. UT – untreated. Values are mean ± SD from 3 independent experiments. **P < 0.01, ****P < 0.0001 (Statistical analysis was performed using unpaired non-parametric two-tailed Mann-Whitney t test). P values compare the percentage of DNA repair between “promoter” and “no promoter” strains for a given time point.

Extended Data Fig. 8 Local transcription enables NER irrespective of Mfd.

Supplementary to Fig. 5. a–h, Depriving the genomic loci of transcription drastically diminished NER irrespective of Mfd. Genomic DNA repair within the insulator was monitored as described in Methods for the lesions generated by 4-NQO (a, e), NFZ (b, f), cisplatin (c, g), or UV-C (d, h). lacZ was induced with IPTG followed by the exposure to drugs or UV radiation. Cells were allowed to recover for the indicated time intervals followed by the isolation of genomic DNA. Lesion density was determined by SLR-qPCR and used to calculate the percentage of repaired lesions. Values are the mean ± SD from 3 independent experiments. i, j, UvrAB recruitment to the UV-damaged DNA strictly depends on local transcription, but not Mfd (compare to Fig. 5l, m). Recruitment of UvrA (i) and UvrB (j) to the lacZ insulator (with or without promoter) was determined by ChIP–qPCR in Δmfd cells as in Fig. 5h. Results are shown as a fold change in the occupancy of UvrAB within the insulator of Δmfd following UV irradiation. **P < 0.01, ****P < 0.0001. Statistical analysis was performed using unpaired non-parametric two-tailed Mann-Whitney t test. Values are the mean  ± SD from 3 independent experiments.

Extended Data Fig. 9 Integrated model of bacterial NER.

See Supplementary Videos 2, 3. a–f, Based on the in vivo and in vitro data presented, we propose a structure-functional model of NER in E. coli in which the elongating RNAP functions as the primary lesion scanner and platform for the assembly of active NER complexes. a, A subpopulation of elongating RNAPs persistently interacts with UvrD1 and UvrA1, as shown in Fig. 2b. The in vivo RNAP pulldowns and XLMS demonstrate that such surveillance pre-TCRCs can form even before the genotoxic stress. b, c, On stalling at a lesion (CPD is marked as “TT”), pre-TCRC recruits UvrD2 to form a helicase competent UvrD dimer. UvrD2 CTD (green hexagon) interacts with RNAP βi4 to stabilize the UvrD dimer. UvrD12 pulls TCRC backward, thereby exposing a CPD to NER enzymes18. ppGpp contributes at this stage by rendering RNAP backtracking-prone12. d, TCRC recruits UvrA2/UvrB to initiate the lesion processing. Although a single UvrB monomer is sufficient for lesion verification and UvrC recruitment28,103,104, the second UvrB molecule may be recruited as well27,102,105. In vitro (Extended Data Fig. 2a–d) and in vivo XLMS (Fig. 2c, d) are consistent with a single UvrB monomer model. This UvrB can interact with the CTD of UvrD2 (Extended Data Fig. 3h–m), thereby displacing UvrD2 from RNAP (Supplementary Video 2). The release of UvrD2 that occurs coincidentally with the UvrA2B recruitment (Fig. 1b, c) supports such a sequence of events in vivo. UvrD2 displacement would abrogate any further UvrD-mediated backtracking. e, The pre-incision TCRC recruits UvrC and releases UvrA2B followed by the NER execution step5,103. f, Once repair has been completed, the backtracked pre-TCRC is promptly recovered by the anti-backtracking factors (GreB, Mfd, and a leading ribosome) to resume elongation. g–k, Role of Mfd in NER (Supplementary Video 3). We propose that the modest contribution of Mfd to NER (Fig. 4a) is due to its ability to terminate multiple queuing ECs in front of TCRCs, thereby helping to “clean up” space between pre-TCRC/TCRCs and DNA lesions at highly expressed genes. g, h, UvrA of pre-TCRC facilitates Mfd recruitment and/or its transition to a processive translocase (Fig. 1d). i, Mfd then translocates forward (downstream of TCRC) to terminate multiple ECs between the TCRC and CPD (red “TT”). This directionality ensures that Mfd preferentially terminates non-TCR complexes, thereby facilitating TCRC access to damage sites. j, TCR proceeds as in (a–f). k, Mfd continues to be recruited even after most repair has been completed (Fig. 1d). These additional Mfd molecules can now also reactivate backtracked complexes, hence the role of Mfd in facilitating transcription recovery post-UV14. This model explains why a delay in NER in Mfd-deficient cells occurs only within the most highly transcribed (most congested) regions and why TCR of less actively transcribed regions is indifferent to, or even compromised by, Mfd activity (for example, Fig. 5c, d)36. It also explains why the overexpression of Mfd is so detrimental to NER36 (Extended Data Fig. 5b): excessive Mfd would prematurely terminate both ECs and TCRCs, thereby abolishing repair. Finally, the model also explains why mfd cells become more sensitive to genotoxic stress in the presence of Rho inhibitor Bcm (Extended Data Fig. 10). Rho, like Mfd, can terminate ECs that obscure the lesion sites from TCRCs41. If both termination factors were inactivated, there is no obvious solution to this problem. l, Structural model for UvrA-mediated Mfd recruitment. (top) E. coli Mfd (colour: cyan) bound to DNA (PDB: 6XEO)106 is shown interacting with UvrA in pre-TCRC by structural alignment to the E. coli UvrA-Mfd complex (PDB: 4DFC)107. UvrA-binding domain of Mfd (colour: blue) is fully unmasked after initial DNA binding, allowing it to be located by pre-TCRC (g–k and Supplementary Video 3). RNAP-binding domain of Mfd (green) is facing downstream to eventually interact with RNAPs stalled/paused ahead of the pre-TCRC and terminate or rescue them from the backtracked state. Illustrative cartoon (bottom). RNA is not shown.

Extended Data Fig. 10 Rho inactivation enhances Mfd sensitivity to UV.

a, Representative efficiencies of colony formation of wild-type (MG1655) and ∆mfd cells on LB agar, LB agar exposed to 40 J/m2 of UV, 25 µM bicyclomycin (Bcm) and 25 µM Bcm with 40 J/m2 of UV. Cells were spotted on LB agar plates in serial tenfold dilutions and incubated at 37 °C for 24 h. b, Data from three independent experiments was used to calculate percent difference in survival between wild-type and ∆mfd cells. Values are means ± SD from 3 independent experiments.

Extended Data Fig. 11 Modelling of pre-TCRC/TCRC with restraints from XLMS.

a, An outline of the automated workflow for cross-link-guided docking. i, ii, Coordinate files for all the E. coli interactors used to build the pre-TCRC and TCRC are prepared using available PDB structures, which were refined using YASARA Structure87 (see Methods). Proteins without available PDB structures were homology-modelled using I-TASSER82,88. XLMS data was converted to the distance restrains compatible with PatchDock. iii, PDB files of receptor and ligand molecules were submitted to PatchDock with their corresponding distance restrains for rigid-body docking. iv, The docking results were validated by examining the cross-link satisfaction using Jwalk91 (see Methods). v, The docked complexes were ranked by the number/fraction of cross-links validated by Jwalk and the average solvent accessible surface distance (SASD). b, An overview of the automated dimer-assembly workflow. Two monomer models (X and Y), previously and separately docked to a common receptor model (R), are combined to generate receptor-dimer models that satisfy the highest number of cross-links between the two monomers. i, ii, Top docking poses for each monomer (R-X and R-Y) are clustered to eliminate redundancies and accelerate subsequent steps. iii, Representative models obtained by clustering each of the two groups are cross-matched to generate combined receptor-dimer coordinate files (R-X-Y), and analysed for cross-links satisfied between X-Y using Jwalk. iv, receptor-dimer models satisfying > 2 cross-links are ranked by number and average distance of satisfied X-Y cross-links for further analysis.

Extended Data Fig. 12 Application of the docking pipeline.

a–d, pre-TCRC modelling. a, PDB files of E. coli ECs were downloaded and prepared by extracting chains corresponding to RNAP subunits and NusA, then refined using YASARA Structure. E. coli UvrA was modelled using the homology template server I-TASSER. b, UvrA was docked to ECs using PatchDock, with the RNAP-UvrA cross-links as distance restraints. c, PDB coordinates file of E. coli UvrD in the apo form was trimmed to the first 640 residues and refined, then docked to the top EC-UvrA (as ranked by cross-link satisfaction) from the previous step. d, EC-UvrA-UvrD complexes generated in the previous step were ranked by RNAP–UvrD cross-link satisfaction and used as receptors to dock UvrD CTD. Results were clustered using ProFit (v.3.1), and finally analysed for the UvrAD DNA-binding regions alignment with the DNA path in the EC. e–g, TCRC modelling. e, Same as a. Docking was repeated using EC-UvrA1 complexes as receptors and additional UvrA-UvrA distance restraints to generate EC-UvrA12 complexes. f, Same as (c). Structure then docked to the top EC-UvrA12 complexes generated in the previous step, as ranked by RNAP-UvrA and UvrA-UvrA cross-link satisfaction. g, Top EC-UvrA12-UvrD complexes generated in the previous step were divided into two groups based on the docked UvrD model (apo vs. closed), and used as input to the dimer-assembly component (Extended Data Fig. 11 and Methods). UvrD poses from the two groups were cross-matched to generate EC-UvrA12-UvrD12 complexes, analysed for UvrD-UvrD cross-link satisfaction and steric clashes, and used as receptors to dock UvrD1 CTD. Results were clustered using ProFit (v.3.1) and analysed for agreement with UvrA-dimer structures and alignment of Uvr DNA-binding regions with the DNA path in the EC. Final complexes were refined with YASARA Structure and re-analysed with Jwalk for cross-link satisfaction.

Extended Data Table 1 Statistics of X-ray crystal structures
Full size table

Supplementary information

Supplementary Information

This file contains Supplementary Discussion, Supplementary Figs. 1–5, Supplementary Tables 1–3, Supplementary References and legends for Supplementary Videos 1–3.

Reporting Summary

Supplementary Video 1

XLMS-driven structural model of TCRC.

Supplementary Video 2

Integrated model of NER in E. coli.

Supplementary Video 3

Mfd functioning in NER.

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Bharati, B.K., Gowder, M., Zheng, F. et al. Crucial role and mechanism of transcription-coupled DNA repair in bacteria. Nature 604, 152–159 (2022). https://doi.org/10.1038/s41586-022-04530-6

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