pairs except ligand 14–ligand 28) or ten (ligand14–ligand 28 pair) experiments.The ligands that were expected to exhibit

positive cooperativity (14 and 28) had a linearstimulus-response relation (Fig. 2G, blue curve);those that were expected to bind independently(14 paired with 26 or 27) yielded nonlinearstimulus-response curves with c values closerto 1 (Fig. 2G, black and gray curves, and tableS1); and those expected to exhibit negative (orless positive) cooperativity (15, 16, 17, or 20paired with 28) yielded a series of curves withincreasingly marked thresholds and decreasinginferred values of c (Fig. 2G, green, yellow, orange,and red curves). Some of the inferred c valueswere higher than expected from simple energeticconsiderations (table S1) (11, 12). Nevertheless,the data demonstrate that negative cooperativitycan, as predicted, imbue a marked threshold anda high degree of ultrasensitivity on the forma-tion of a ternary complex between a receptor andtwo high-affinity ligands.Another feature of the classical sequential

models (models 1 and 2) is that receptor acti-vation increases linearly with the total con-centration of receptor (eqs. S7 and S8). This isnot true, however, for model 4, which con-siders ligand depletion. In the high-affinitylimit, the response is predicted to be biphasic.As the receptor concentration increases, theoutput initially increases, peaks when the sto-ichiometric ratio of receptor to ligand is 1:2,and decreases thereafter (Fig. 3, A to C) (13).The narrowness of the response peak dependsupon the cooperativity of the binding; positivecooperativity makes the fall-off in output at highreceptor concentrations occur gradually, and neg-ative cooperativitymakes the fall-off more abrupt(Fig. 3, A to C). This phenomenon is conceptual-ly related to the prozone effect, which was dis-covered in early studies of antigen-antibodyinteractions (14, 15); to the phenomenon of tran-scriptional squelching (16–18); and to the inhibi-tion of protein kinase signaling by overexpressionof scaffold proteins (19–22). Essentially, the re-ceptor is behaving like a bivalent adaptor, andhigh concentrations of the receptor drive the for-mation of binary ligand-receptor complexes at theexpense of the full ternary complex.We therefore determined whether complex for-

mation was a biphasic function of receptor con-centration, again using the oligonucleotide ligandsshown in Fig. 2C. The formation of the full ternarycomplex was biphasic, in good agreement withtheory (Fig. 3, D to G). The ligands that were ex-pected to exhibit the highest degrees of negativecooperativity produced the sharpest biphasic peaks,and, as shown in table S1, the inferred cooper-ativity values agreed reasonably well with thoseobtained from the threshold data in Fig. 2.Our results show that for the high-affinity

interaction of a multimeric receptor with adepletable ligand, marked thresholds and highdegrees of ultrasensitivity can arise as a resultof negative cooperativity in the binding reac-tions. Such thresholds can allow a system tofilter out small stimuli and yet respond deci-

sively to suprathreshold stimuli. Moreover, thethresholds and ultrasensitivity that arise throughthis mechanism can be critical elements for theproduction of more complex systems-level be-haviors, such as bistability and oscillations (23).In addition, multimeric receptors can exhibit abiphasic response to changes in the concentra-tion of the receptor, and this biphasic response issharpest when strong negative cooperativity is ineffect. These basic aspects of the regulation ofmultimeric proteins may be important both forunderstanding the behavior of natural regula-tory systems and for designing synthetic ones.

REFERENCES AND NOTES

1. D. E. Koshland Jr., G. Némethy, D. Filmer, Biochemistry 5,365–385 (1966).

2. A. Levitzki, D. E. Koshland Jr., Proc. Natl. Acad. Sci. U.S.A. 62,1121–1128 (1969).

3. D. E. Koshland Jr., A.Goldbeter, J. B. Stock,Science217, 220–225 (1982).4. M. A. Savageau, Curr. Top. Cell. Regul. 6, 63–130 (1972).5. J. E. Ferrell Jr., Trends Biochem. Sci. 21, 460–466 (1996).6. N. E. Buchler, M. Louis, J. Mol. Biol. 384, 1106–1119 (2008).7. J. E. Ferrell Jr., S. H. Ha, Trends Biochem. Sci. 39, 556–569 (2014).8. N. Jullien, J. P. Herman, Biotechniques 51, 267–269 (2011).9. E. T. Kool, Annu. Rev. Biophys. Biomol. Struct. 30, 1–22 (2001).10. P. Yakovchuk, E. Protozanova, M. D. Frank-Kamenetskii,

Nucleic Acids Res. 34, 564–574 (2006).11. R. Owczarzy, Y. You, C. L. Groth, A. V. Tataurov, Biochemistry

50, 9352–9367 (2011).12. IDT-Biophysics, “DNA thermodynamics and hybridization,”

2015; http://biophysics.idtdna.com/.13. S. H. Ha, S. Y. Kim, J. E. Ferrell Jr., Cell Rep. 14, 1408–1421 (2016).14. D. Bray, S. Lay, Proc. Natl. Acad. Sci. U.S.A. 94, 13493–13498 (1997).

15. M. Heidelberger, F. E. Kendall, J. Exp. Med. 50, 809–823 (1929).16. M. A. Cahill, W. H. Ernst, R. Janknecht, A. Nordheim, FEBS Lett.

344, 105–108 (1994).17. S. Natesan, V. M. Rivera, E. Molinari, M. Gilman, Nature 390,

349–350 (1997).18. R. Prywes, H. Zhu, Nucleic Acids Res. 20, 513–520 (1992).19. A. Levchenko, J. Bruck, P. W. Sternberg, Proc. Natl. Acad. Sci.

U.S.A. 97, 5818–5823 (2000).20. M. Dickens et al., Science 277, 693–696 (1997).21. A. M. Cacace et al., Mol. Cell. Biol. 19, 229–240 (1999).22. N. R. Michaud et al., Proc. Natl. Acad. Sci. U.S.A. 94, 12792–12796

(1997).23. J. E. Ferrell Jr., S. H. Ha, Trends Biochem. Sci. 39, 612–618 (2014).24. A. Goldbeter, D. E. Koshland Jr., Proc. Natl. Acad. Sci. U.S.A. 78,

6840–6844 (1981).

ACKNOWLEDGMENTS

We thank D. Herschlag and R. Owczarzy for helpful discussions andG. Anderson, B. Beltran, X. Cheng, and T. H. Lee for comments onthe paper. This work was supported in part by grants from the U.S.NIH (R01 GM046383 and P50 GM107615) and from the NationalResearch Foundation of Korea (NRF-2011-357-C00151 and2012M3A9B9036680). S.H.H. and J.E.F. jointly designed thestudies, made the figures, and wrote the paper. S.H.H. carried outthe experiments and J.E.F. did the theoretical work.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/352/6288/990/suppl/DC1Materials and MethodsSupplementary TextFigs. S1 to S3Table S1Reference (25)

6 October 2015; accepted 7 April 2016Published online 12 May 201610.1126/science.aad5937

DNA REPAIR

ppGpp couples transcriptionto DNA repair in E. coliVenu Kamarthapu,1,2 Vitaly Epshtein,1 Bradley Benjamin,1 Sergey Proshkin,3

Alexander Mironov,3 Michael Cashel,4 Evgeny Nudler1,2*

The small molecule alarmone (p)ppGpp mediates bacterial adaptation to nutrientdeprivation by altering the initiation properties of RNA polymerase (RNAP). ppGpp isgenerated in Escherichia coli by two related enzymes, RelA and SpoT.We show that ppGppis robustly, but transiently, induced in response to DNA damage and is required forefficient nucleotide excision DNA repair (NER). This explains why relA-spoT-deficientcells are sensitive to diverse genotoxic agents and ultraviolet radiation, whereas ppGppinduction renders them more resistant to such challenges. The mechanism of DNAprotection by ppGpp involves promotion of UvrD-mediated RNAP backtracking. Byrendering RNAP backtracking-prone, ppGpp couples transcription to DNA repair andprompts transitions between repair and recovery states.

Bacteria respond to starvation by rapidlyaccumulating guanosine-3′,5′-(bis)pyro-phosphate (ppGpp). This results in theinhibition of ribosomal and transfer RNAtranscription and metabolic transition,

known as the stringent response, which allowsbacteria to conserve energy and adapt to adverseconditions (1, 2). The global reprogramming ofgene expression is a consequence of ppGpp bind-ing to RNA polymerase (RNAP), which causesdestabilization of open promoter complexes(3, 4). ppGpp acts synergistically with the tran-scription factor DksA, which also interacts with

RNAP (5, 6). Evidence suggests ppGpp also func-tions independently in preserving genomic in-tegrity (7–10). Here, we demonstrate that ppGppis crucial for the process of nucleotide excisionDNA repair (NER).The majority of bulky DNA lesions are elimi-

nated by NER (11). Escherichia coli resistance tohelix-distorting mutagens, such as ultraviolet(UV) radiation, 4-nitroquinoline-1-oxide (4NQO),and nitrofurazone (NFZ), depends on the intra-cellular level of ppGpp (8, 10) (Fig. 1A and fig. S1).Cells lacking relA and spoT (ppGpp0) were sen-sitive to these stressors, whereas relA256 spoT203

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cells, which produced excess ppGpp under nor-mal exponential growth conditions, were moreresistant than the parent control (Fig. 1A and fig.S1). ppGpp was transiently induced in responseto these challenges (Fig. 1B). Its level increasedmore than 20 times within the first 15 min ofgenotoxic stress and then returned to basal levelswithin 45 min. Because DNA lesions caused byUV, 4NQO, and NFZ are eliminated predomi-nantly by NER (12), we examined the potentialrole of ppGpp in NER.RNAP facilitates the recognition of DNA

damage by NER enzymes through transcription-coupled DNA repair (TCR) (13). ppGpp is a mod-ulator of RNAP activity and, therefore, couldsupport NER by functioning in TCR. To testthis, we compared the ability of wild-type andppGpp-deficient cells to remove the UV-inducedcyclobutane pyrimidine dimers (CPDs) fromthe individual DNA strands of the lac operon(Fig. 1C and fig. S2). Wild-type isopropyl-b-D-thiogalactopyranoside (IPTG)–induced cells re-paired CPDs in the transcribed strand of lacoperon more rapidly than in the nontranscribedstrand (14) (Fig. 1C and fig. S2). In contrast, ppGpp0

cells displayed the same slow rate of repair on bothstrands, which was similar to the rate of repair ofthe nontranscribed strand in wild-type cells (Fig.1C and fig. S2). Thus, ppGpp is important for TCR.In the “forward translocation” TCR pathway,

the translocase Mfd binds to the upstream edgeof stalledRNAP (15) and pushes it forward againstthe lesion, which terminates transcription (16, 17).Mfd then recruits UvrA to the site of damage(16) to initiate NER. The “backtracking” pathwayrelies on the 3′-5′ helicase UvrD, which bindsRNAPandpulls it backward, away from the lesionsite, which exposes the lesions to NER enzymes(18). To determinewhich of the twoTCRpathwaysrequires ppGpp, we combined the relA spoT dele-tions with either uvrD or mfd (Fig. 2A). The sen-sitivity of Dmfd DrelAspoT to 4NQO, NZF, or UVwas approximately equal to that of the sum of thesensitivity of individual Dmfd and ppGpp0mutantstrains. An additive effect of Mfd and ppGpp indi-cates that the two factors act in different repairpathways. In contrast, the DuvrD DrelAspoT mu-tant was as sensitive to genotoxic agents and UVas DuvrD alone (Fig. 2A and fig. S3). The epistaticrelation between UvrD and ppGpp suggests thatthey act in the same, backtracking-mediated TCRpathway.Transcript cleavage factors GreA and GreB

interfere with UvrD-mediated TCR, as they com-pete with UvrD-mediated backtracking (18). In-activation of these antibacktracking factors greatlysuppressed the sensitivity of ppGpp0 cells tobeing killed by genotoxic agents and UV (Fig.

2B and fig. S4); from this, we argue that ppGppacts as a probacktracking factor inUvrD-mediatedTCR. In bacteria, transcription and translation arecoupled. The leading ribosome directly controlsthe rate of transcription elongation by preventingRNAP backtracking (19) and, thereby, also inter-feres with UvrD-mediated TCR (18). Analogous tothe situation with Gre-deficiency, we show thatslowing ribosome translocation with a sublethaldose of chloramphenicol (Cm) rendered ppGpp0

cells more resistant to genotoxic chemicals andUV (Fig. 2C and fig. S5).DksA has been shown to promote ppGpp ac-

tivities in vivo and in vitro (5, 6). Accordingly,DksA-deficient cells were sensitive to 4NQO, NFZ,and mitomycin C, whereas inactivation of GreAfully suppressed this sensitivity (8) (Fig. 2Dand fig.S6). Moreover, DksA overexpression suppressedthe sensitivity to genotoxic stress in the absence ofppGpp (fig. S7). Collectively, these in vivo resultsargue that ppGpp, in conjunction with DksA, pro-motes UvrD-mediated TCR by facilitating RNAPbacktracking.To test whether ppGpp assists UvrD in pro-

moting RNAP backtracking, we examined theeffect of ppGpp andUvrD on transcription elonga-tion in a reconstituted single-round runoff assay(Fig. 3A and fig. S8). UvrD forced RNAP to stall atmany sites so that only a small fraction of tran-scription complexes reached the end of the tem-plate to form a full-length runoff product (18). Themajority of observedpaused or arrested complexeswere the result of UvrD-induced backtracking,

hence, their sensitivity to GreB (Fig. 3B and tableS1). Addition of 100 mM ppGpp, a concentrationcorresponding to the cellular level of ppGppduring the stringent response, greatly stimu-lated UvrD-mediated backtracking: Less than20% of RNAP molecules traversed the middleof the template in the presence of ppGpp to-gether with UvrD (Fig. 3A, lane 6), whereasmore than 50% of RNAPs did so in the pres-ence of UvrD alone (Fig. 3A, lane 3). DksA +ppGpp further promoted the probacktrackingactivity of UvrD (Fig. 3A, lanes 7 to 9). DksAalone potentiated UvrD-mediated backtrack-ing only slightly (Fig. 3A, lanes 10 to 12).The structural model of the ppGpp-RNAP

complex suggests that ppGpp facilitates par-tial opening of the RNAP clamp (4), whichmay render RNAP prone to backtracking. Totest this prediction, we monitored the front edgeof a stable elongation complex (EC) stalled at po-sition +32 (EC32) by probing it with exonucleaseIII (Fig. 3C and fig. S8). The footprinting of EC32showed that RNAP was almost evenly distributedbetween post and pretranslocated states (lane 3).ppGpp shifts the equilibrium toward the pre-translocated state (lane 4). Longer incubationwith exonuclease III resulted in progressive back-tracking, which was more prominent in the pre-sence of ppGpp than in its absence (lanes 5 and 6).To confirm that ppGpp also facilitates RNAP

backtracking in vivo, we used a plasmid (p1EC)in which the Lac repressor bound to its opera-tor site blocks an isolated EC ~70 nucleotides

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1Department of Biochemistry and Molecular Pharmacology,New York University School of Medicine, New York, NY 10016,USA. 2Howard Hughes Medical Institute, New York UniversitySchool of Medicine, New York, NY 10016, USA. 3EngelhardtInstitute of Molecular Biology, Russian Academy of Science,Moscow 119991, Russia. 4Division of Developmental Biology,Intramural Research Program, Eunice Kennedy Shriver NationalInstitute of Child Health and Human Development, NationalInstitutes of Health, Bethesda, MD 20892, USA.*Corresponding author. Email: evgeny.nudler@nyumc.org

Fig. 1. ppGpp is required for genotoxic stress survival and TCR. (A) ppGpp0 cells (DrelA DspoT) orcells producing excessive amounts of ppGpp (relA256spoT203) (29) were treated with 4NQO, NFZ, orUV. Data from three independent experiments are presented as the means ± SEM; **P < 0.01; *P < 0.05.Representative efficiencies of colony formation are shown in fig. S1. (B) MG1655 cells were treated with4NQO or NFZ for the indicated time. ppGpp was detected by thin-layer chromatography. The same area(rectangle) in each lane was used for ppGpp quantification. Values are the means (±SEM) of three inde-pendent experiments. (C) Strand-specific repair of the lacZ operon (6.6 kilobases of Apa I–Sst II fragment)was measured at 0, 10, 20, 30, and 40 min after UV irradiation (50 J/m2). Data from three independentexperiments (fig. S2) are presented as the means ± SEM.T, transcribed strand; NT, nontranscribed strand.

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downstream of the transcription start site (Fig.3D) (20). To monitor the effect of ppGpp andUvrD on the positioning of the halted EC, weperformed in situ DNA footprinting using thesingle strand–specific probe, chloroacetaldehyde(CAA). Cells transformed with the plasmid pEC1alone or pEC1 together with the UvrD over-expression plasmid (pUvrD) were treated withserine hydroxamate (SHX) to induce the strin-gent response (fig. S8). ppGpp induction causedthe roadblocked EC to backtrack over a longerdistance (Fig. 3D, compare lanes 3 and 4): CAAreactive sites were more prominent upstream ofthe footprint, and the reactivity of the down-stream margin of the bubble was diminished.Backtracking was substantially more extensivein the SHX-treated cells that overexpressed UvrD(lane 5). SHX failed to promote backtrackingin UvrD-deficient cells (fig. S9). Thus, as shown

in vitro, ppGpp also facilitates UvrD-mediatedRNAP backtracking in vivo. Consistently, SHX-pretreated cells are more resistant to genotoxicstress (fig. S10).A stringent RNAP allele, rpoB*35, carries a

point mutation [in which glutamine replacesHis1244 (H1244Q)] in the beta subunit that mimicsthe presence of ppGpp (7). rpoB*35 suppressedthe sensitivity of ppGpp0 cells to genotoxic agentsand UV (8) (Fig. 2E and fig. S11). In the absenceof UvrD, however, cells harboring rpoB*35 wereunable to effectively suppress their sensitivity toDNA-damaging agents and were almost as sen-sitive as uvrD– cells (fig. S12), which suggestedthat rpoB*35 acted via UvrD-mediated TCR. Totest this directly, we examined the effect of UvrDand ppGpp on rpoB*35 RNAP in vitro. RpoB*35was purified and used in a single-round assay(Fig. 3A). In the absence of UvrD, the RpoB*35

enzyme elongated slightly faster than wild-typeenzyme (21) and was less responsive to certainstrong pauses (compare Fig. 3A, lanes 1 and 13)(21). However, RpoB*35 becamemuchmore proneto backtracking in the presence of UvrD (Fig. 3A,lanes 14 and 15). The extent of UvrD-mediatedbacktracking by RpoB*35 was comparable tothat of wild-type enzyme in the presence ofppGpp and DksA (compare Fig. 3A, lanes 8 and 9,as well as 14 and 15). Moreover, addition of ppGpp(with or without DksA) to the RpoB*35 reactionhad no further effect on UvrD-mediated back-tracking (lanes 17 and 18). We thus conclude thatRpoB*35 mimics the effect of ppGpp on wild-typeRNAP. These results provide direct support for theUvrD- and ppGpp-mediated TCR and explainhow ppGpp couples transcription to DNA repair.Here, we implicate ppGpp as an important

component of UvrD-mediated TCR (fig. S13).

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Fig. 2. ppGpp contributes to the UvrD-mediated (backtracking) TCRpathway. (A) MG1655 cells lacking Mfd, UvrD, or RelA and SpoTor combi-nations of Mfd-RelA-SpoTand UvrD-RelA-SpoTwere exposed to the indicatedamounts of 4NQO and NFZ. Data from three independent experiments arepresented as the means ± SEM; **P < 0.01; *P < 0.05; ns, nonsignificant.Representative efficiencies of colony formation are shown in fig. S3. (B) In-activating antibacktracking factors GreA and GreB suppress ppGpp0 sensi-tivity to 4NQO, NFZ, and UV. Data from three independent experiments arepresented as the means ± SEM (P < 0.01). Representative efficiencies ofcolony formation are shown in fig. S4. (C) Slowing ribosomal translocation

with a sublethal concentration of chloramphenicol suppresses ppGpp0 sen-sitivity to 4NQO or UV. Data from three independent experiments are presentedas the means ± SEM (P < 0.01). Representative efficiencies of colony formationare shown in fig. S5. (D) Inactivating GreA suppresses the sensitivity of DksA-deficient cells to genotoxic stress caused by mitomycin C, 4NQO, or NFZ. Datafrom three independent experiments are presented as the means ± SEM (P <0.01). Representative efficiencies of colony formation are shown in fig. S6.(E) The rpoB*35 allele suppresses ppGpp0 sensitivity to 4NQO, NFZ, and UV.Data from three independent experiments are presented as the means ± SEM(P < 0.01). Representative efficiencies of colony formation are shown in fig. S11.

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Our in vivo and in vitro data indicate thatppGpp and UvrD act together to promote back-tracking and that this probacktracking activityaccounts for most of their phenotypes withrespect to DNA damage. Modeling suggeststhat RNAP undergoes a conformational changeupon binding ppGpp, which widens its “claw-pincers” (4) and renders it prone to backtracking(22). In the absence of stress, the E. coli cellcontains ~3 × 103 molecules of UvrD (23), atan approximately 1:1 ratio with RNAP. Consid-ering the high affinity of UvrD for RNAP (Kd =35 nM), most of the UvrD should be sequesteredby RNAP under normal growth conditions. How-ever, for UvrD to act as a helicase capable ofbacktracking, it must form a dimer (18, 24). Suchdimers are not stable (Kd ≈ 470 nM) (25). As theinduction of UvrD during the SOS response isonly two- to threefold (26), UvrD dimerizationis expected to occur only intermittently duringstress. Thus, by lowering the energy barrier nec-essary to cause backtracking, ppGpp widens thecritical window of opportunity for UvrD to actin TCR. DksA contributes substantially to UvrD-and/or ppGpp-mediated TCR (figs. S6 and S7),because it stabilizes RNAP in the backtracking-prone state imposed by ppGpp (Fig. 3A) (4) andcompetes with antibacktracking Gre factors forthe same binding site on RNAP (6).Codirectional collisions between the repli-

cation fork and backtracked RNAP often lead

to DNA double-strand breaks (21). However,because ppGpp accumulates only during thecritical phase of genotoxic stress (Fig. 1B), whenit also inhibits replication (9, 27), such detri-mental collisions are effectively avoided. Thetimely decline of ppGpp triggers the recoveryprocess (fig. S13), which relies on the concertedaction of antibacktracking factors—GreAB, activeribosomes, and Mfd—that compete with pro-backtracking UvrD (Fig. 2 and figs. S4, S5, S14,and S15) (18, 19, 28). Thus, ppGpp not only helpsto activate UvrD-TCR but also ensures that theprocess is deactivated promptly so that trans-cription may recover and that conflicts withreplication are avoided.

REFERENCES AND NOTES

1. L. U. Magnusson, A. Farewell, T. Nyström, Trends Microbiol. 13,236–242 (2005).

2. K. Potrykus, H. Murphy, N. Philippe, M. Cashel, Environ.Microbiol. 13, 563–575 (2011).

3. M. M. Barker, T. Gaal, C. A. Josaitis, R. L. Gourse, J. Mol. Biol.305, 673–688 (2001).

4. Y. Zuo, Y. Wang, T. A. Steitz, Mol. Cell 50, 430–436 (2013).5. B. J. Paul et al., Cell 118, 311–322 (2004).6. A. Parshin et al.,Proc. Natl. Acad. Sci. U.S.A. 112, E6862–E6871 (2015).7. P. McGlynn, R. G. Lloyd, Cell 101, 35–45 (2000).8. B. W. Trautinger, R. P. Jaktaji, E. Rusakova, R. G. Lloyd,

Mol. Cell 19, 247–258 (2005).9. J. DeNapoli, A. K. Tehranchi, J. D. Wang, Mol. Microbiol. 88,

93–104 (2013).10. K. E. Madison et al., Mol. Microbiol. 92, 28–46 (2014).11. J. T. Reardon, A. Sancar, Prog. Nucleic Acid Res. Mol. Biol. 79,

183–235 (2005).

12. D. P. Batty, R. D. Wood, Gene 241, 193–204 (2000).13. V. Kamarthapu, E. Nudler, Curr. Opin. Microbiol. 24, 15–20 (2015).14. I. Mellon, P. C. Hanawalt, Nature 342, 95–98 (1989).15. L. F. Westblade et al., Nucleic Acids Res. 38, 8357–8369 (2010).16. C. P. Selby, A. Sancar, Science 260, 53–58 (1993).17. J. S. Park, M. T. Marr, J. W. Roberts, Cell 109, 757–767 (2002).18. V. Epshtein et al., Nature 505, 372–377 (2014).19. S. Proshkin et al., Science 328, 504–508 (2010).20. V. Epshtein et al., EMBO J. 22, 4719–4727 (2003).21. D. Dutta et al., Cell 146, 533–543 (2011).22. S. Sekine, Y. Murayama, V. Svetlov, E. Nudler, S. Yokoyama,

Mol. Cell 57, 408–421 (2015).23. H. M. Arthur, P. B. Eastlake, Gene 25, 309–316 (1983).24. N. K. Maluf et al., J. Mol. Biol. 325, 913–935 (2003).25. N. K. Maluf, T. M. Lohman, J. Mol. Biol. 325, 889–912 (2003).26. E. C. Siegel, Mol. Gen. Genet. 191, 397–400 (1983).27. G. Schreiber, E. Z. Ron, G. Glaser, Curr. Microbiol. 30, 27–32 (1995).28. B. J. Schalow, C. T. Courcelle, J. Courcelle, J. Bacteriol. 194,

2637–2645 (2012).29. E. Sarubbi, K. E. Rudd, M. Cashel,Mol. Gen. Genet. 213, 214–222 (1988).

ACKNOWLEDGMENTS

We thank R. Lloyd for N4849 and N4235 strains and T. Artemyev for hiscontribution. This work was supported by the Russian ScienceFoundation grant 14-50-00060 and the Ministry of Education andScience of the Russian Federation grant 14.Z50.31.0014 (S.P. andA.M.),the National Institute of Child Health and Human Development, NIHIntramural Program (M.C.), NIH grant R01 GM107329, and by theHoward Hughes Medical Institute (E.N.).

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/352/6288/993/suppl/DC1Materials and MethodsFigs. S1 to S15Tables S1 and S2References (30–41)

21 October 2015; accepted 7 April 201610.1126/science.aad6945

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Fig. 3. ppGpp promotes UvrD-mediated RNAPbacktracking in vitro and in vivo. (A) EC20 wasformed by wild-type RNAP or RpoB*35 (lanes 13to 18) at the T7A1 DNA template and then chased inthe presence of specified amounts of UvrD. ppGppand/or DksA were added to the chase reaction asindicated.The probacktracking activity of UvrD wasassessed as a ratio (%) between the total amountsof RNA products located above and below the ar-bitrary midsection line indicated by red asterisks.Mean values ± SEM (P < 0.05) from three indepen-dent experiments are shown in table S1. (B) EC20(lane 1) immobilized on Co++-beads was chasedwith (lanes 3 to 6) or without UvrD+ppGpp (lane 2),followed by washing (lane 4). GreB without (lanes 5and 6) or with nucleoside triphosphates (lane 6;second chase) was added. Red lines connect exem-plary corresponding RNA from arrested ECs beforeand after GreB treatment (lanes 4 and 5) to illustratetranscript cleavage. The red asterisk denotes thesame position as in (A). (C) The top and bottom pa-nels resolve the same probes to show protection ofthe 32P–end-labeled nontemplate DNA strand ofEC32 from Exo III digestion in the presence or ab-sence of ppGpp. Blue boxes correspond to the areasof Exo III footprinting at the front edge of EC32. Theweight of the red lines illustrates the intensity of thefootprint signal. Bands corresponding to the pre- andposttranslocated states, as well as backtrackedspecies, are indicated. The extent of backtracking(%) was measured as the ratio between all backtracked signals and the total RNA signal in the corresponding lane. (D) The p1EC constructs (left) (19) andprimer extension analyses (right). CAA modifications on the nontemplate strand of p1EC (lanes 2 and 3), p1EC in the presence of the ppGpp inducer, SHX (lane4), or p1EC1+SHX+pUvrD (lane 5). The lac operator (Lac) and transcription bubble are indicated. Red lines show the position of blocked EC. Experimentalworkflows for A, B, C, and D are shown in fig. S8.

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E. colippGpp couples transcription to DNA repair in

NudlerVenu Kamarthapu, Vitaly Epshtein, Bradley Benjamin, Sergey Proshkin, Alexander Mironov, Michael Cashel and Evgeny

DOI: 10.1126/science.aad6945 (6288), 993-996.352Science 

, this issue p. 993Sciencereplication, it also avoids dangerous collisions between the replication fork and backtracked RNA polymerase.ppGpp helps backtrack the RNA polymerase away from the DNA damage to facilitate repair. Through inhibiting DNA

pathway.ppGpp is also essential for DNA repair. ppGpp couples transcription elongation to the nucleotide excision repair show thatet al.This ''stringent response'' helps them conserve energy and survive adverse conditions. Kamarthapu

-(bis)pyrophosphate (ppGpp) shuts down transcription in bacteria that are starving.′,5′The alarmone guanosine-3A starvation survival signal fights DNA damage

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