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UvrD facilitates DNA repair by pulling RNA polymerase backwards

Abstract

UvrD helicase is required for nucleotide excision repair, although its role in this process is not well defined. Here we show that Escherichia coli UvrD binds RNA polymerase during transcription elongation and, using its helicase/translocase activity, forces RNA polymerase to slide backward along DNA. By inducing backtracking, UvrD exposes DNA lesions shielded by blocked RNA polymerase, allowing nucleotide excision repair enzymes to gain access to sites of damage. Our results establish UvrD as a bona fide transcription elongation factor that contributes to genomic integrity by resolving conflicts between transcription and DNA repair complexes. Furthermore, we show that the elongation factor NusA cooperates with UvrD in coupling transcription to DNA repair by promoting backtracking and recruiting nucleotide excision repair enzymes to exposed lesions. Because backtracking is a shared feature of all cellular RNA polymerases, we propose that this mechanism enables RNA polymerases to function as global DNA damage scanners in bacteria and eukaryotes.

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Figure 1: UvrD promotes RNAP backtracking.
Figure 2: RNAP backtracking facilitates NER.
Figure 3: Anti-backtracking factors obstruct UvrD activity in NER.
Figure 4: Mapping UvrD interactions with the elongation complex.
Figure 5: UvrD and NusA cooperate in backtracking-mediated NER.

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Acknowledgements

We thank D. Jeruzalmi for materials. This work was supported by the Russian Foundation for Basic Research (A.M.) and the NIH, BGRF, Dynasty foundation and by the Howard Hughes Medical Institute (E.N.).

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Contributions

V.E., V.K., K.M., V.S., B.U., S.P. and A.M. conducted the experimental work, discussed the results and commented on the manuscript. E.N. designed the study and wrote the paper.

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Correspondence to Evgeny Nudler.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 UvrD binds RNAP.

a, A sample list of RNAP-bound proteins from exponentially grown E. coli. Abundance is based on emPAI score15. b, UvrD + 6His–RNAP, UvrD, or 6His–RNAP were affinity-purified on nickel beads and electrophoresed alongside pure UvrD, for reference. The asterisk indicates UvrD. c, RNAP–UvrD complex isolated by size-exclusion chromatography. UvrD–RNAP were mixed 3:1, and run over a Superdex 200 10/300GL column. RNAP core (red) and UvrD (green) chromatograms are overlaid for comparison. The inlaid polyacrylamide gel displays fractions taken from each chromatographic peak.

Extended Data Figure 2 UvrD promotes long-range backtracking.

Biotinylated RNAP was used to prepare the startup EC11 immobilizing on beads. It was walked to position 39, followed by incubation with UvrD, washed (to remove UvrD and NTPs) and then treated with GreB for the indicated times. Numbers on the right indicate the size of 5′-labelled RNAs.

Extended Data Figure 3 Thymine dimer in the template strand blocks the elongation complex.

Schematic diagram of the T7A1 promoter template shows the position of CPD (red). Both control (no TT) and CPD-bearing template strands were radiolabelled at their 5′ ends (top band). The sequencing gel also shows the radiolabelled RNA products before and after chase of EC11. The EC was completely halted by CPD (indicated by red TT). On the control template it formed the runoff products.

Extended Data Figure 4 Effect of greAB, mfd and ribosome inactivation on uvrD sensitivity to DNA damaging agents and ultraviolet.

a, Representative efficiencies of colony formation of wild-type (MG1655) and mutant E. coli cells in the presence of the indicated amounts of mitomycin C, 4NQO and cisplatin. Cells were spotted on LB agar plates in serial tenfold dilutions and incubated at 30 °C for 24 h. b, Representative efficiencies of colony formation of wild-type and ΔuvrD cells in the presence of the indicated amounts of mitomycin, 4NQO and chloramphenicol. Cells were spotted on LB agar plates in serial tenfold dilutions and incubated at 30 °C for 24 h. c, Representative efficiencies of colony formation of wild-type (MG1655) and mutant E. coli cells after ultraviolet irradiation. Cells were spotted on LB agar plates in serial tenfold dilutions and incubated at 30 °C for 24 h. d, Representative efficiencies of colony formation of wild-type and mutant E. coli cells after ultraviolet irradiation in the presence of a sublethal dose of chloramphenicol. Cells were spotted on LB agar plates in serial tenfold dilutions and incubated at 30 °C for 24 h.

Extended Data Figure 5 UvrD inactivation suppresses temperature sensitivity of greAB cells.

E. coli strains were streaked on LB agar plates and incubated at the indicated temperatures for 24 h.

Extended Data Figure 6 UvrD–RNAP crosslinks.

a, Three inter-protein crosslinks indicate that UvrD binds near the β flap on RNAP. UvrD (grey, PDB accession code 2IS4) crosslinks to RNAP (PDB accession code 4IGC) at three distinct positions that span the β (pale yellow) and β′ (light blue) subunits of RNAP. The non-template strand (blue, PDB accession code 4G7O) is indicated for reference. Crosslinked lysines are colored magenta and pairs are connected with a black line. b, MS2 spectrum of a representative crosslinked pair (β′ K40)–(UvrD K448). The peptide sequences with crosslinked lysine residues are shown (top right). Observed peptide backbone cleavage is indicated and b- and y-type fragment ions are labelled in the spectrum. The m/z tolerances of fragment ions are presented in the inset below the spectra. Spectra were annotated using pLabel50.

Extended Data Figure 7 GreB inactivation suppresses nusA sensitivity to genotoxic chemicals and ultraviolet.

a, Representative efficiencies of colony formation of wild-type (MG1655) and mutant E. coli cells in the presence of indicated amounts of NFZ and 4NQO. Cells were spotted on LB agar plates in serial tenfold dilutions and incubated at 30 °C for 24 h. b, Data from three independent experiments are presented as the mean ± s.e.m.; **P < 0.01. c, Representative efficiencies of colony formation of MDS42 and mutant E. coli cells in the presence of indicated amounts of mitomycin, 4NQO, NFZ and after ultraviolet irradiation. Cells were spotted on LB agar plates in serial tenfold dilutions and incubated at 30 °C for 24 h.

Extended Data Figure 8 Deletion of mfd partially suppresses uvrD sensitivity to mitomycin C.

a, Representative efficiencies of colony formation of wild-type (MG1655) and mutant E. coli cells in the presence of indicated amounts of mitomycin C. Cells were spotted on LB agar plates in serial tenfold dilutions and incubated at 30 °C for 24 h. b, Data from three independent experiments are presented as the mean ±s.e.m.; **P < 0.01.

Extended Data Figure 9 Transcriptional arrest as a function of UvrD concentration.

a, A representative chase experiment demonstrating multiple transcriptional arrests as a function of UvrD concentration. b, Data from three independent experiments are plotted as the mean ± s.e.m. Arrest efficiency (%) was calculated as a fraction of all arrested complexes in relation to the full-length runoff.

Extended Data Table 1 Escherichia coli strains used in this study

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Epshtein, V., Kamarthapu, V., McGary, K. et al. UvrD facilitates DNA repair by pulling RNA polymerase backwards. Nature 505, 372–377 (2014). https://doi.org/10.1038/nature12928

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