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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Reardon, J. T. & Sancar, A. Nucleotide excision repair. Prog. Nucleic Acid Res. Mol. Biol. 79, 183–235 (2005)
Van Houten, B. & McCullough, A. Nucleotide excision repair in E. coli. Ann. NY Acad. Sci. 726, 236–251 (1994)
Ganesan, A., Spivak, G. & Hanawalt, P. C. Transcription-coupled DNA repair in prokaryotes. Prog. Mol. Biol. Transl. Sci. 110, 25–40 (2012)
Truglio, J. J., Croteau, D. L., Van Houten, B. & Kisker, C. Prokaryotic nucleotide excision repair: the UvrABC system. Chem. Rev. 106, 233–252 (2006)
Mellon, I. & Hanawalt, P. C. Induction of the Escherichia coli lactose operon selectively increases repair of its transcribed DNA strand. Nature 342, 95–98 (1989)
Gaillard, H. & Aguilera, A. Transcription coupled repair at the interface between transcription elongation and mRNP biogenesis. Biochim. Biophys. Acta 1829, 141–150 (2013)
Deaconescu, A. M. et al. Structural basis for bacterial transcription-coupled DNA repair. Cell 124, 507–520 (2006)
Park, J. S., Marr, M. T. & Roberts, J. W. E. coli transcription repair coupling factor (Mfd protein) rescues arrested complexes by promoting forward translocation. Cell 109, 757–767 (2002)
Savery, N. Prioritizing the repair of DNA damage that is encountered by RNA polymerase. Transcription 2, 168–172 (2011)
Selby, C. P. & Sancar, A. Molecular mechanism of transcription–repair coupling. Science 260, 53–58 (1993)
Kumura, K. & Sekiguchi, M. Identification of the uvrD gene product of Escherichia coli as DNA helicase II and its induction by DNA-damaging agents. J. Biol. Chem. 259, 1560–1565 (1984)
Lee, J. Y. & Yang, W. UvrD helicase unwinds DNA one base pair at a time by a two-part power stroke. Cell 127, 1349–1360 (2006)
Matson, S. W. & George, J. W. DNA helicase II of Escherichia coli. Characterization of the single-stranded DNA-dependent NTPase and helicase activities. J. Biol. Chem. 262, 2066–2076 (1987)
Cohen, S. E. et al. Roles for the transcription elongation factor NusA in both DNA repair and damage tolerance pathways in Escherichia coli. Proc. Natl Acad. Sci. USA 107, 15517–15522 (2010)
Ishihama, Y. et al. Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol. Cell. Proteomics 4, 1265–1272 (2005)
Nudler, E., Gusarov, I. & Bar-Nahum, G. Methods of walking with the RNA polymerase. Methods Enzymol. 371, 160–169 (2003)
Nudler, E. RNA polymerase backtracking in gene regulation and genome instability. Cell 149, 1438–1445 (2012)
Borukhov, S., Sagitov, V. & Goldfarb, A. Transcript cleavage factors from E. coli. Cell 72, 459–466 (1993)
Nudler, E., Mustaev, A., Lukhtanov, E. & Goldfarb, A. The RNA–DNA hybrid maintains the register of transcription by preventing backtracking of RNA polymerase. Cell 89, 33–41 (1997)
Brosh, R. M., Jr & Matson, S. W. Mutations in motif II of Escherichia coli DNA helicase II render the enzyme nonfunctional in both mismatch repair and excision repair with differential effects on the unwinding reaction. J. Bacteriol. 177, 5612–5621 (1995)
Proshkin, S., Rahmouni, A. R., Mironov, A. & Nudler, E. Cooperation between translating ribosomes and RNA polymerase in transcription elongation. Science 328, 504–508 (2010)
Donahue, B. A., Yin, S., Taylor, J. S., Reines, D. & Hanawalt, P. C. Transcript cleavage by RNA polymerase II arrested by a cyclobutane pyrimidine dimer in the DNA template. Proc. Natl Acad. Sci. USA 91, 8502–8506 (1994)
Selby, C. P., Drapkin, R., Reinberg, D. & Sancar, A. RNA polymerase II stalled at a thymine dimer: footprint and effect on excision repair. Nucleic Acids Res. 25, 787–793 (1997)
Selby, C. P. & Sancar, A. Transcription preferentially inhibits nucleotide excision repair of the template DNA strand in vitro. J. Biol. Chem. 265, 21330–21336 (1990)
Manelyte, L., Kim, Y. I., Smith, A. J., Smith, R. M. & Savery, N. J. Regulation and rate enhancement during transcription-coupled DNA repair. Mol. Cell 40, 714–724 (2010)
Batty, D. P. & Wood, R. D. Damage recognition in nucleotide excision repair of DNA. Gene 241, 193–204 (2000)
Doudney, C. O. & Rinaldi, C. N. Chloramphenicol-promoted increase in resistance to UV damage in Escherichia coli B/r WP2 trpE65: development of the capacity for successful repair of otherwise mutagenic or lethal lesions in DNA. Mutat. Res. 143, 29–34 (1985)
Hanawalt, P. C. The U.V. sensitivity of bacteria: its relation to the DNA replication cycle. Photochem. Photobiol. 5, 1–12 (1966)
Tomko, E. J. et al. 5′-Single-stranded/duplex DNA junctions are loading sites for E. coli UvrD translocase. EMBO J. 29, 3826–3839 (2010)
Korzheva, N. et al. A structural model of transcription elongation. Science 289, 619–625 (2000)
Toulokhonov, I., Artsimovitch, I. & Landick, R. Allosteric control of RNA polymerase by a site that contacts nascent RNA hairpins. Science 292, 730–733 (2001)
Bar-Nahum, G. et al. A ratchet mechanism of transcription elongation and its control. Cell 120, 183–193 (2005)
Ahn, B. A physical interaction of UvrD with nucleotide excision repair protein UvrB. Mol. Cells 10, 592–597 (2000)
Manelyte, L. et al. The unstructured C-terminal extension of UvrD interacts with UvrB, but is dispensable for nucleotide excision repair. DNA Repair 8, 1300–1310 (2009)
Tornaletti, S. Transcription arrest at DNA damage sites. Mutat. Res. 577, 131–145 (2005)
Trautinger, B. W., Jaktaji, R. P., Rusakova, E. & Lloyd, R. G. RNA polymerase modulators and DNA repair activities resolve conflicts between DNA replication and transcription. Mol. Cell 19, 247–258 (2005)
Arthur, H. M. & Eastlake, P. B. Transcriptional control of the uvrD gene of Escherichia coli. Gene 25, 309–316 (1983)
Maluf, N. K., Fischer, C. J. & Lohman, T. M. A dimer of Escherichia coli UvrD is the active form of the helicase in vitro. J. Mol. Biol. 325, 913–935 (2003)
Dutta, D., Shatalin, K., Epshtein, V., Gottesman, M. E. & Nudler, E. Linking RNA polymerase backtracking to genome instability in E. coli. Cell 146, 533–543 (2011)
Witkin, E. M. Mutation and the repair of radiation damage in bacteria. Radiat. Res. 6, (suppl.). 30–53 (1966)
Schalow, B. J., Courcelle, C. T. & Courcelle, J. Mfd is required for rapid recovery of transcription following UV-induced DNA damage but not oxidative DNA damage in Escherichia coli. J. Bacteriol. 194, 2637–2645 (2012)
Brueckner, F. & Cramer, P. DNA photodamage recognition by RNA polymerase II. FEBS Lett. 581, 2757–2760 (2007)
Churchman, L. S. & Weissman, J. S. Nascent transcript sequencing visualizes transcription at nucleotide resolution. Nature 469, 368–373 (2011)
Compe, E. & Egly, J. M. TFIIH: when transcription met DNA repair. Nature Rev. Mol. Cell Biol. 13, 343–354 (2012)
Drapkin, R. et al. Dual role of TFIIH in DNA excision repair and in transcription by RNA polymerase II. Nature 368, 769–772 (1994)
Yang, B. et al. Identification of cross-linked peptides from complex samples. Nature Methods 9, 904–906 (2012)
Murakami, K. S. X-ray crystal structure of Escherichia coli RNA polymerase σ70 holoenzyme. J. Biol. Chem. 288, 9126–9134 (2013)
Runyon, G. T., Wong, I. & Lohman, T. M. Overexpression, purification, DNA binding, and dimerization of the Escherichia coli uvrD gene product (helicase II). Biochemistry 32, 602–612 (1993)
Pakotiprapha, D., Samuels, M., Shen, K., Hu, J. H. & Jeruzalmi, D. Structure and mechanism of the UvrA-UvrB DNA damage sensor. Nature Struct. Mol. Biol. 19, 291–298 (2012)
Li, D. et al. pFind: a novel database-searching software system for automated peptide and protein identification via tandem mass spectrometry. Bioinformatics 21, 3049–3050 (2005)
Lutz, R. & Bujard, H. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res. 25, 1203–1210 (1997)
Epshtein, V., Toulme, F., Rahmouni, A. R., Borukhov, S. & Nudler, E. Transcription through the roadblocks: the role of RNA polymerase cooperation. EMBO J. 22, 4719–4727 (2003)
Pósfai, G. et al. Emergent properties of reduced-genome Escherichia coli. Science 312, 1044–1046 (2006)
Cardinale, C. J. et al. Termination factor Rho and its cofactors NusA and NusG silence foreign DNA in E. coli. Science 320, 935–938 (2008)
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.).
The authors declare no competing financial interests.
Extended data figures and tables
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.
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.
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.
E. coli strains were streaked on LB agar plates and incubated at the indicated temperatures for 24 h.
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.
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.
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.
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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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