Translesion DNA polymerases remodel the replisomeand alter the speed of the replicative helicaseChiara Indiania, Lance D. Langstona, Olga Yurievaa, Myron F. Goodmanb, and Mike O’Donnella,1

aLaboratory of DNA Replication, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, Box 228, New York, NY 10065;and bDepartments of Biological Sciences and Chemistry, University of Southern California, Los Angeles, CA 90089

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2006.

Contributed by Mike O’Donnell, February 11, 2009 (sent for review January 22, 2009)

All cells contain specialized translesion DNA polymerases thatreplicate past sites of DNA damage. We find that Escherichia colitranslesion DNA polymerase II (Pol II) and polymerase IV (Pol IV)function with DnaB helicase and regulate its rate of unwinding,slowing it to as little as 1 bp/s. Furthermore, Pol II and Pol IV freelyexchange with the polymerase III (Pol III) replicase on the �-clampand function with DnaB helicase to form alternative replisomes,even before Pol III stalls at a lesion. DNA damage-induced levels ofPol II and Pol IV dominate the clamp, slowing the helicase andstably maintaining the architecture of the replication machinerywhile keeping the fork moving. We propose that these dynamicactions provide additional time for normal excision repair oflesions before the replication fork reaches them and also enablethe appropriate translesion polymerase to sample each lesion as itis encountered.

DNA repair � DNA replication � replication fork � replisome sliding clamp

Chromosomes are duplicated by replisome machines contain-ing helicase, primase, and DNA polymerase activites (1).

Replicative DNA polymerases are tethered to DNA by ring-shaped sliding clamp proteins. The Escherichia coli polymeraseIII (Pol III) holoenzyme replicase contains 10 different subunitsconsisting of a clamp loader that binds 2 molecules of Pol III,each tethered to DNA by the �-sliding clamp for processiveleading and lagging strand synthesis (2). Pol III also binds tightlyto the hexameric DnaB helicase that encircles the lagging strandtemplate. The Pol III-to-DnaB interaction is mediated by the�-subunit within Pol III (3). This tight connection couples Pol IIImotion to DnaB unwinding and increases the rate of unwindingby DnaB helicase from a basal level of 35 bp/s to a rate in excessof 500 bp/s (3). These tight connections of Pol III to DnaB andthe �-clamp provide the replisome with sufficient stability, inprinciple, to replicate the entire genome. In practice, however,replication of long chromosomal DNA is an uneven path with avariety of obstacles along the way, including protein blocks anddamaged templates.

In the presence of high levels of DNA damage, �40 geneproducts are induced by the SOS response that act to restoregenomic integrity and assure cell survival (4). Among theSOS-induced gene products are 3 translesion DNA polymerases,Pols II, IV, and V (encoded by polB, dinB, and umuCD,respectively), that perform potentially mutagenic DNA synthesisacross template lesions (5). Pols IV and V are members of theY family of error-prone DNA polymerases; they lack a proof-reading 3�–5� exonuclease (6, 7). Pol II is a B-family polymerase;it has high fidelity and contains proofreading activity (8). Pols IIand IV are present during normal cell growth and may beinvolved in repairing low levels of DNA damage that occurroutinely in the cell (9–11). Pol V, however, is closely associatedwith mutagenesis and is not detectable in cells under normalconditions (7, 12, 13).

Pol II and Pol IV are among the first genes that are up-regulated in the SOS response, whereas the levels of Pol V are

very slow to rise, peaking 45 min after SOS induction (4, 9, 11,12). In vitro studies have shown that these DNA polymerasesfunction with the �-clamp for lesion bypass and trade places on� with a stalled Poll III replicase, as originally demonstrated inbacteriophage T4 (14–17). Once the lesion is bypassed, thehigh-fidelity Pol III can switch back on the �-clamp to reestablishthe fast moving E. coli replisome (650 nt per s) (14, 17). In thisview of translesion synthesis, the TLS polymerase only occupiesthe �-clamp during the short time interval of lesion bypass.

It seems unlikely that TLS polymerases would be able toreplace Pol III in the context of a moving replisome, becauseTLS polymerases are very slow and have low fidelity. Thesecharacteristics are inconsistent with the rapid speed and highfidelity required for chromosome replication. It is possible thatTLS polymerases are excluded from replisomes by the tightconnection between Pol III and the DnaB helicase via the�-subunit. Additional insurance against TLS polymerase entryinto the replisome is the known action of DnaB helicase when itbecomes uncoupled from Pol III, whereupon it continues foronly �1 kb before DnaB stops unwinding (18–20). Presumably,DnaB dissociates from DNA when it is uncoupled from leading-strand Pol III action. Once DnaB dissociates, it cannot get backonto DNA without specialized helicase-loading factors, andreplication fork progression is halted. Therefore, even if a TLSpolymerase enters the replisome, the subsequent loss of Pol IIIinteraction with DnaB may result in DnaB dissociation, prevent-ing further progression of the fork.

Fork breakdown upon takeover by Pol IV is supported byrecent studies that show that overexpression of Pol IV in theabsence of DNA damage inhibits cell growth in Bacillus subtilisand E. coli (21, 22). The authors of the E. coli study (21) note thatinduction of Pol IV stops replication abruptly and kills the cell,suggesting that Pol IV entry into the replisome is catastrophic.Interestingly, these observations did not require the clampbinding residues of Pol IV, suggesting that Pol IV blocksreplication by a process distinct from TLS polymerase switchingon a clamp. However, Pol IV was overexpressed to abnormallyhigh levels in that study. Thus, the mechanism of the replicationblock by Pol IV was suggested to be separate from the physio-logical action of the enzyme. Lower levels of Pol IV expressionappeared to decrease replication without cell killing, suggestingPol IV may slow a replication fork, but this hypothesis could notbe confirmed and was not tested in vitro.

Author contributions: C.I., L.D.L., and M.O.D. designed research; C.I., L.D.L., and O.Y.performed research; M.F.G. contributed new reagents/analytic tools; C.I., L.D.L., M.F.G.,and M.O.D. analyzed data; and C.I., L.D.L., and M.O.D. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.

See Commentary on page 6027.

1To whom correspondence should be addressed. E-mail: odonnel@mail.rockefeller.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0901403106/DCSupplemental.

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In the current study we examine the effects of normal andinduced levels of Pol II and Pol IV on moving replisomesreconstituted in vitro. We find that these TLS polymerases cangain access to the moving Pol III replisome despite the strong PolIII–�–DnaB connection. Surprisingly, the fork does not breakdown. Instead the TLS polymerases function with DnaB to formreplisomes that move more slowly than the intrinsic rate ofDnaB, with the rate of helicase unwinding adjusting to the speedof the polymerase at the fork. At the levels of Pol II and Pol IVpresent in undamaged cells, the Pol III replisome is unaffected,whereas at levels of Pol II and Pol IV that correspond to theirconcentrations in DNA damage-induced cells, the TLS poly-merases dominate the replication fork and slow it down. Ex-pression of Pol II and Pol IV in vivo in the absence of DNAdamage also slows DNA replication and depends on TLSpolymerase interaction with the �-clamp. Slow replication forkadvance in the context of DNA damage makes biological senseas it buys time for normal DNA repair, decreasing collisionevents of the fork with DNA lesions.

ResultsPol II and Pol IV Form Alternative Replisomes with the �-Clamp andDnaB Helicase. To investigate possible alternative roles for Pol IIand Pol IV beyond translesion synthesis, we examined theirability to form a functional replisome with the �-clamp andDnaB helicase in the absence of Pol III. For these studies, weused a minicircle replication fork substrate consisting of asynthetic 100-mer circular duplex with a 5� T40 ssDNA tail (20,23). Each strand of the synthetic minicircle substrate containsonly 3 nt: dG, dC, dA on the leading strand template, and dG,dC, dT on the lagging stand template. Hence, leading and laggingstrands can be specifically labeled in separate reactions usingeither [�32P] dTTP or [�32P] dATP, respectively.

The replication machinery at the fork was assembled byloading DnaB on the 5� dT40 tail of the minicircle DNA in the

absence of ssDNA-binding protein (SSB), then the �-clamp wasloaded onto DNA by using the clamp loader followed by theaddition of Pol II or Pol IV in the presence of only dCTP anddGTP to prevent fork movement. After 5 min, replication wasinitiated upon adding dATP, dTTP, SSB, DnaG primase, andthe 4 rNTPs (see Fig. 1A). In these assays, dissociation of DnaBfrom DNA will halt the fork because SSB is present and the assaydoes not contain DnaB-reloading factors. Replication fork pro-gression is followed by analysis of timed aliquots in an alkalineagarose gel. The results are compared with similar reactionsperformed in the absence of DnaB helicase or the absence of theclamp and clamp loader.

Surprisingly, the analysis shows that Pol II and Pol IV functionwith DnaB and the �-clamp, because the presence of both ofthese factors is required to synthesize long DNA chains (Fig. 1B and C, lanes 1–6). DNA synthesis continues for at least 20 min,indicating that the DnaB helicase acts processively and is main-tained the entire time. The rates of Pol II and Pol IV forkprogression are �10 nucleotides (ntd)/s and 1 ntd/s, respectively(Fig. S1), significantly slower than Pol III replisomes under thesame conditions and consistent with the slower synthetic rates ofPol II and Pol IV compared with Pol III (24, 25). The ability ofPol II and Pol IV to function with DnaB in the absence of PolIII indicates that replisomes containing these TLS polymerasesremain intact rather than undergo replication fork breakdown.These results also suggest that the speed of a replicative helicasecan be limited by the rate of synthesis of the polymerase travelingalong behind it. Lagging strand products are also observed byusing [�32P] dATP as the radiolabel as shown in Fig. S2. Wepresume that leading and lagging strand synthesis is uncoupledunder these conditions, as further described in Discussion.

Both Pol II and Pol IV are present in normal cells and aregreatly up-regulated during the SOS response. As shown in Fig.1D, the rate of DNA synthesis in the presence of Pol II and PolIV (Center and Right) was slower than that of Pol II alone (Left),

Fig. 1. Pol II and Pol IV form alternative replisomes with DnaB helicase and the � clamp. (A) Minicircle DNA was incubated with DnaB, then TLS polymerase(5 pmol), clamp loader, clamp and dCTP, dGTP for 5 min. Replication was initiated by addition of SSB, DnaG primase, dATP, dTTP, [�32P]dTTP, and the 4 rNTPs.Newly-synthesized leading strand DNA is in red, and lagging strand is in blue. As controls, either DnaB or clamp/clamp loader were excluded from the reaction.(B and C) Time courses of Pol II (B) or Pol IV (C) in the presence of DnaB, the �-clamp, and the clamp loader (lanes 1–6), in the absence of clamp and clamp loader(lanes 7–12), and in the absence of DnaB (lanes 13–18). Products were analyzed in alkaline agarose gels. (D) Pol II (15 pmol) and Pol IV [5 pmol (Center) or 7 pmol(Right)] were added simultaneously to the reaction. (Left) The gel shows synthesis by Pol II in the absence of Pol IV for comparison.

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suggesting that both polymerases have access to the fork andalternate in taking control of the growing DNA ends in adynamic fashion. If the 2 polymerases did not repeatedly tradeplaces at the fork, 2 distinct products would be observed thatcorrespond to the rates of Pol II- and Pol IV-driven replicationforks. We confirmed that Pol II acts in a distributive manner atthe replication fork by showing that the speed of Pol II repli-somes depends on the Pol II concentration (Fig. S3).

Pol III Switches with Pol II and Pol IV to Produce a Rapid Replisome.The experiments of Fig. 1 indicate that DnaB is a component ofthe Pol II and Pol IV replisomes and presumably stimulatessynthesis by unwinding DNA. However, DnaB is not known tofunction with other DNA polymerases, nor is DnaB known tounwind DNA as slowly as observed in these alternative repli-somes. Hence, it remained possible that DnaB helicase is actingin some other capacity to stimulate synthesis on the minicirclereplication fork substrate. If DnaB is truly functional andencircles DNA in the alternative replisomes, then Pol III shouldbe capable of displacing the TLS polymerase with resumption ofthe rapid unwinding characteristic of the coupled DnaB–Pol IIIreplisome. To examine this issue and provide further evidencethat replisomes containing Pol II or Pol IV are fully functionaland have not broken down, we performed a series of pulse–chaseexperiments by adding Pol III to a prelabeled replication forkcontaining a moving Pol II or Pol IV (see Fig. 2A). Pol II or PolIV was first assembled with DnaB, the sliding clamp, and clamploader in a 5-min preincubation, and then DNA synthesis wasinitiated for 7 min (Pol II) or 15 min (Pol IV) in the presence of[�32P] dTTP (pulse). After this labeling reaction, a large excess

of unlabeled dNTPs (chase) was added to prevent incorporationof additional [�32P] dTTP. Then, Pol III holoenzyme lacking the�-clamp (referred to as Pol III*) was added. If Pol III* takescontrol of the replication fork, the radiolabel incorporated by PolII or Pol IV during the pulse period will quickly shift to a highermolecular mass during the chase period because of the rapid rateof synthesis by Pol III*-�. As shown in Fig. 2B, Pol II (Left)synthesized �3 kb during the 7-min pulse of [�32P] dTTP, andPol IV (Right) synthesized �1 kb during a 15-min pulse (lanes 1,5, and 9), consistent with the results of Fig. 1. Addition of a largeexcess of cold dNTPs had no effect on the rate of synthesis byPol II or Pol IV in the absence of added Pol III* (Fig. 2B, lanes2, 6, and 10), but when Pol III* was added immediately after thechase, the pulse-labeled band shifted dramatically higher, in atime-dependent manner (Fig. 2B, lanes 3, 7, and 11). As a controlfor the effectiveness of the chase, Pol III* was added toreplication forks assembled without Pol II or Pol IV and showedno detectable incorporation of [�32P] dTTP (Fig. 2B, lanes 4, 8,and 12).

This pulse–chase experiment confirms that the DnaB helicaseis fully functional within the alternative Pol II and Pol IVreplisomes and that the basic architecture of the machinery atthe replication fork is maintained by the specialized DNApolymerases despite their slow speed compared with Pol III*-�.Hence, DnaB is stable on DNA in a functional state, and its rateof DNA unwinding is determined by the speed of the DNApolymerase that functions with it. We also conclude that Pol III*takeover of Pol II and Pol IV replisomes is rapid and efficient.

Pol II and Pol IV Recruit the Clamp and Helicase from a Moving Pol IIIReplisome. Our previous studies with singly-primed ssDNA cir-cles showed that when Pol III holoenzyme was stalled at a primedsite by nucleotide omission Pol IV takes control of the primerterminus even at low concentrations of Pol IV, but when Pol IIIis engaged in active DNA synthesis, much higher levels of Pol IVare required to take over the primed site (14). However, at areplication fork the Pol III holoenzyme has several additionalconnections that may anchor it to DNA and block entry of otherDNA polymerases. Specifically, at a replication fork Pol IIIconnects to 2 �-clamps on both the leading and lagging strandsthrough its twin polymerase structure. Furthermore, Pol IIIholoenzyme also connects to the DnaB helicase via the �-subunit(26). These Pol III-specific connections may prevent takeover ofa Pol III replisome by Pol II or Pol IV.

Based on the results of Figs. 1 and 2, one would predict thatif Pol II or Pol IV can recruit the �-clamp from a Pol IIIreplisome, the result would be a functional, but slow, alternativereplisome. To examine the ability of TLS polymerases to takeover a moving replication fork, increasing concentrations of PolII and Pol IV were added to a moving Pol III-based replisomein the minicircle replication fork assay along with [�32P]dTTP(leading strand) or [�32P]dATP (lagging strand), as illustrated inFig. 3A. Timed aliquots were collected and analyzed in analkaline agarose gel. The first 4 lanes in Fig. 3B show the longleading-strand products typically formed by the fast-moving PolIII replisome, as reported (20). Reactions were then analyzed inthe presence of increasing amounts of Pol II (Fig. 3B Left, lanes5–20) or Pol IV (Fig. 3B Right, lanes 5–20). The results indicatethat both Pol II and Pol IV interfere with the accumulation oflong leading-strand products generated by Pol III in a dose-dependent manner. Inspection of the gel clearly shows that PolIV is more efficient at inhibiting the replisome than Pol II;rolling circle replication was reduced �50% upon addition of 0.5pmol of Pol IV (Fig. 3B Right, lanes 9–12), whereas 2 pmol of PolII was required for a 40% decrease of DNA synthesis (Fig. 3BLeft, lanes 13–16). Lagging-strand replication was reduced to thesame extent as leading-strand synthesis (Fig. S4).

All 5 E. coli polymerases function with the clamp and bind �

Fig. 2. Pol III regains control from a moving Pol II- or Pol IV-based replisome.(A) The Pol II- or Pol IV-based replisome was assembled as in Fig. 1A. After a7-min pulse (Pol II) or 15-min pulse (Pol IV), a 100-fold excess of unlabeleddNTPs (chase) was added 10 s before Pol III (1 pmol), and reactions werequenched at different times after the addition of Pol III. Radiolabeled DNAsynthesized by the TLS polymerase during the pulse period is shown in red, andDNA synthesized by Pol III during the chase period is in blue. (B) DNA synthesisby Pol II (Left) or Pol IV (Right) without Pol III and cold dNTPs (lanes 1, 5, and9), with cold dNTPs (lanes 2, 6, and 10), and with Pol III and cold dNTPs (lanes3, 7, and 11). Control experiments where Pol III was added to an unoccupiedfork after addition of cold dNTPs are shown in lanes 4, 8, and 12.

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through a conserved consensus sequence (27). Pol II and Pol IVbind to � via the C-terminal 5 or 6 amino acid residues,respectively (24, 28). To determine whether Pol II and Pol IVfunction with � in takeover of Pol III-based replisomes, weexpressed and purified mutant versions of Pol II and Pol IV thatlack their respective C-terminal �-binding residues and testedthem in the minicircle replication assay outlined in Fig. 3A. Forthese experiments, 5 pmol of either WT or mutant Pol II or PolIV was added 10 s after initiation of replication by Pol III. Asexpected for �-dependent function, the C-terminal mutant PolII and Pol IV were no longer capable of inhibiting Pol III-basedreplisomes in the minicircle replication assay (compare WTlanes 7–12 vs. �C-term lanes 13–18 for Pol II, Fig. 3C Left, andPol IV, Fig. 3C Right). These results support the conclusion thatPol II and Pol IV take control of the replisome from Pol III bycompetitive binding to the clamp and slow the fork. As a control,we determined that the mutant Pol II and Pol IV retained �70%of the activity of the corresponding WT polymerase in gap-fillingassays that do not require �.

Pol III is connected to the DnaB helicase at the replication

fork by the �-subunit of the clamp loader, and this connection isessential for rapid, processive rolling circle replication by PolIII*. Because Pol III is poorly processive in the absence of �, wewere not able to determine whether � plays a role in the takeoverof a Pol III replisome by Pol II or Pol IV. We did, however,determine that � is not required for rolling circle replication byPol II (Fig. S5), suggesting that the role played by � in stimulatingDnaB-dependent replication might be unique to Pol III, perhapsby making Pol III more processive in the context of the repli-cation fork rather than by directly stimulating unwinding byDnaB.

Pol II and Pol IV Remodel the Pol III-Based Replisome and SlowReplication Fork Advance. The results of Fig. 3 indicate that wheneither Pol II or Pol IV is added to a Pol III-based replisome, thereplisome is remodeled to include the TLS polymerase, leadingto a slower moving fork that does not break down but remainsintact. Nonetheless, it remains possible that Pol II and Pol IVsimply inhibit the replication reaction without taking control ofthe replication fork or stop the fork and cause it to break down.Hence, we designed the experiments illustrated in the scheme ofFig. 4A to determine whether the replication fork continues toslowly advance after the addition of Pol II or Pol IV. Because PolIII-based replisomes quickly produce replication products thatapproach the resolution limit of alkaline gels, we examined veryshort time points after addition of the TLS polymerase. In thecase of Pol II, it is clear that the fork does not break down; forkprogression continues but at a slower rate (Fig. 4B Left). Pol IVappears to stop the replication fork (Fig. 4B Right), but consid-ering that the Pol IV fork only moves 1 ntd/s it would only extendDNA by 120 nt at the final time point of this experiment. Thisshort extension cannot be detected considering that the Pol III

Fig. 3. Pol II and Pol IV takeover of Pol III-based replisomes. (A) The replisomeis assembled on DNA as in Fig. 1, except Pol III* is used instead of Pol II or PolIV. After 10 s, different amounts of TLS polymerase (pink) are added togetherwith [�32P]dTTP to label leading-strand DNA. Newly synthesized DNA is in blue(lagging) and red (leading). (B) Time courses of Pol III replication in thepresence of 0 pmol (lanes 1–4), 0.1 pmol (lanes 5–8), 0.5 pmol (lanes 9–12), 2pmol (lanes 13–16) and 5 pmol (lanes 17–20) of Pol II (Left) or Pol IV (Right).Timed aliquots of leading strands were analyzed in alkaline agarose gels(lagging strand analysis is in Fig. S4). (C) Five picomoles of WT Pol II or Pol IV(lanes 7–12) or Pol II/IV �C-term (lanes 13–18) were added to a moving Pol IIIon a minicircle substrate. Leading-strand synthesis is shown (Pol III only is inlanes 1–6).

Fig. 4. Pol II and Pol IV slow down the replication fork. (A) The Pol III-basedreplisome was assembled as described in Fig. 3A. DNA synthesized by Pol III isin blue, and DNA synthesized by the TLS polymerase is in red. (B) After 25 s (0time point, lanes 1 and 9) a high concentration of Pol II (27 pmol; Left) or PolIV (35 pmol; Right) was added, and aliquots were collected (lanes 2–8). Timecourses of Pol III alone (lanes 9–16) are shown for comparison. The dotted redline highlights the difference in DNA chain length at 120 s with and withoutadded Pol II.

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replisome has already moved nearly 10 kb by the time Pol IV isadded. Based on the results of Fig. 1C, showing that Pol IVfunctions with DnaB, and Fig. 2B, showing that Pol IV repli-somes are competent for resumption of synthesis by Pol III*, wepresume that takeover of the fork by Pol IV causes the fork toslow dramatically rather than break down.

Induction of Pol II and Pol IV in the Cell Reduces ChromosomeReplication in the Absence of DNA Damage. The in vitro reactionsof Figs. 3 and 4 predict that high levels of Pol II and/or Pol IV,as found in the early stages of the SOS response, take over thereplisome and slow down the rate of chromosome replication invivo. To test this prediction, the genes encoding Pol II and PolIV were cloned into a plasmid under control of the induciblearabinose promoter, and the incorporation of [3H] thymidineduring replication was measured before and after induction (seeFig. 5A). The amounts of arabinose used for induction yielded1,200 molecules of Pol II per cell and 2,500 molecules of Pol IVper cell, within 2- to 3-fold the levels present in the cell duringthe SOS response (9, 10). The results of Fig. 5B indicate thatinduction of Pol II is accompanied by a 55% decrease in DNAreplication, and induction of Pol IV decreases DNA synthesis by�99% (Fig. 5C). The more severe decrease in DNA synthesisupon overexpression of Pol IV compared with Pol II correlateswell with the results of the in vitro assays of Fig. 3. Controlexperiments using the same plasmid, but without the Pol II or PolIV genes, showed that the vector does not interfere withchromosomal replication.

To test whether the replication inhibition observed uponexpression of Pol II and Pol IV in vivo depends on interactionof the 2 specialized polymerases with the �-clamp, genes en-coding Pol II and Pol IV mutants that lack the C-terminal�-interacting residues were cloned into the arabinose inducibleplasmid and tested for [3H] thymidine incorporation in thepresence and absence of arabinose. The mutant polymeraseswere found to have no measurable effect on DNA synthesis. Theresults in Fig. 5 D and E indicate that Pol II and Pol IV interactwith � to reduce intracellular replication, consistent with the invitro results of Fig. 3C.

Although we were unable to distinguish whether DNA syn-thesis in vivo was slowed or stopped by overexpression of Pol IIand Pol IV, the results of Fig. 5 are remarkably consistent withthe in vitro experiments of Fig. 3, suggesting that the reconsti-tuted rolling circle replication system can be mimicked by Pol IIand Pol IV overexpression in living cells. Despite the goodagreement of the in vivo and in vitro data, we cannot eliminatethe possibility that differences in sulA inhibition of cell growthcould reflect a differential induction of the SOS response whenWT Pol II or Pol IV are overexpressed, and thereby mightaccount, at least in part, for the differences in DNA synthesis(Fig. 5 B and C). It is interesting to note that UV irradiation ofPol II mutant cells delays recovery of replication restart (29),which may be caused, in part, by more efficient takeover by PolIV in the absence of Pol II (i.e., Pol IV is the more potent of the2 polymerases in slowing the fork). However, a subsequent studydid not observe a defect in the rate of recovery after UVirradiation of either Pol II or Pol IV mutant cells (30), suggestingthat the rate of recovery depends on the kinetics of the repairprocesses themselves and not on whether replication proceedsslowly while repair is occurring behind the fork.

DiscussionThis article demonstrates that E. coli translesion DNA poly-merases Pol II and Pol IV form alternative replisomes withDnaB helicase (Fig. 1) and maintain the architecture of thereplication machinery for eventual resumption of synthesis byPol III (Fig. 2). Whether overexpressed in the cell or studied invitro, high levels of Pol II and Pol IV clearly interfere withongoing processive synthesis by Pol III (Figs. 3 and 5), by slowingrather than stopping the replisome (Fig. 4), and this inhibition ofPol III replication depends on the interaction of Pol II and PolIV with the �-clamp both in vitro (Fig. 3) and in vivo (Fig. 5).On the basis of these results, we conclude that high levels of PolII and Pol IV recruit both the �-clamp and DnaB helicase fromPol III at a moving replication fork during the SOS response.Surprisingly, these TLS polymerases modulate the rate of theDnaB helicase and slow the replication fork without causing it tobreak down.

DnaB as a Regulated Motor. DnaB is not known to function withother polymerases besides Pol III, yet Pol II and Pol IV slow therate of DnaB unwinding, suggesting they may connect to DnaBand regulate helicase speed. A reinterpretation of previousstudies suggests another explanation. Specifically, the rate ofDnaB unwinding may simply be determined by the rate of anyDNA polymerase that acts on the unwound ssDNA product asillustrated in bacteriophage T4 and T7 systems (43, 44). The 35bp/s rate of DnaB unwinding observed in an earlier study wasdetermined by monitoring chain extension by Pol III in theabsence of the �-subunit (3). Because �, which binds DnaB, wasnot present it was thought that the observed rate of unwindingwas the intrinsic rate of DnaB. Instead, the observed unwindingrate may represent the rate of DnaB unwinding when function-ing with Pol III core. The same study also observed a 35 bp/s rateby the primosome, which contains DnaB and other proteins,including DnA helicase (3). The current study shows that Pol II

Fig. 5. Expression of Pol II and Pol IV in vivo reduces chromosomal DNAsynthesis. The rate of DNA synthesis is expressed as incorporation of [3H]thy-midine over a 3-min interval starting 45 min after overexpression of the TLSpolymerase by the addition of arabinose. (A) Scheme of the experiment asdetailed in Experimental Procedures. (B) Arabinose-induced expression of PolII WT. (C) Pol IV WT. (D) Pol II �C-term. (E) Pol IV �C-term. Pol II �C-term andPol IV �C-term are impaired in their ability to bind the clamp. For B–E, 3Hincorporation was adjusted to account for different cell densities of unin-duced and induced cultures.

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and Pol IV, which are slower than Pol III, function with DnaBto move replication forks at rates of 10 and 1 bp/s, respectively.If the intrinsic rate of DnaB unwinding is truly 35 bp/s, thecurrent study indicates that Pol II and Pol IV slow DnaB. Thehighest rate of DnaB unwinding is observed by using Pol IIIholoenzyme containing � (�500 bp/s).

In light of these 4 different rates of unwinding, ranging�500-fold, we propose that the DnaB unwinding rate is coupledto DNA synthetic rate, even in the absence of a direct couplingfactor like �. This observation suggests that DnaB acts as a‘‘regulated motor’’ that must be coupled to some other activity,like DNA polymerization, to promote efficient unwinding (43,44). A replicative helicase acting as a regulated motor will ensurethat unwinding is tightly coordinated with DNA synthesis, afavorable property for replisome action. Otherwise, ssDNA willbe generated that, besides being vulnerable to nucleases andstrand breaks, leads to the SOS response.

Logic of Slower Alternative Replisomes in the Face of DNA Damage.The concept of switching between Pol III and TLS polymeraseshas been considered an ephemeral phenomenon, where a stalledreplicative polymerase briefly yields to the TLS polymerase onlyfor bypass of the lesion, and Pol III immediately retakes controlof the growing DNA end to resume processive replication. Theresults shown here compel a reconsideration of this notion,because high levels of Pol II and Pol IV, as found in the earlystages of the SOS response, clearly take over replication by PolIII to form slower replisomes that function not only with theclamp but also with the replicative DnaB helicase. Unlike Pol IIIreplicase that contains 2 Pol III cores connected to a clamploader, Pol II and Pol IV are not known to dimerize or connectto other proteins besides �. Hence, we presume that leading- andlagging-strand synthesis in TLS replisomes are constitutivelyuncoupled, with no need to hypothesize a DNA trombone loop(illustrated in Fig. 6). We propose that �-clamps are assembledon lagging strand RNA primers by the �-complex, and perhapsit may be the reason for production of both � and � in cells.Nevertheless, it remains possible that TLS polymerases dimerizeor connect to other proteins in the context of a replication fork.

The Pol II and Pol IV replisomes are highly stable andtherefore are probably the active form during the entire SOSresponse. These replisomes are also highly dynamic. We dem-

onstrate here that all 3 DNA polymerases (Pols II, III, and IV)freely exchange with one another while preserving the DNA-bound helicase and sliding clamps. We presume the averageresidence time of any particular polymerase at the fork is definedby the relative amounts of the different DNA polymerases in thecell, their processivity, and their affinity for the sliding clamp andhelicase. Likewise, the speed of the replication fork will be afunction of the residence time of each DNA polymerase and itsintrinsic rate of DNA polymerization. As the intracellular con-centration of TLS polymerases decreases, the population time ofPol III at the replication fork will increase, with a correspondingincrease in replication fork speed.

What would be the purpose of establishing slower replicationforks in the face of DNA damage? We propose an additional rolefor SOS-induced Pol II and Pol IV besides their ability to bypasslesions. Specifically that TLS polymerases slow the fork, whichmay give more time for repair of DNA damage by the excisionrepair system, which can only function on dsDNA before areplication fork collides with a lesion. Of course, sometimes thereplisome will encounter a lesion, and TLS synthesis will bypassit, producing a mutation. Nevertheless, slower moving forksshould lower the frequency of these events. Pol II and Pol IV areinduced early in the SOS damage response and thereforereplication fork control by these polymerases is expected to bespecific to the first phase (�45 min) of SOS induction before thehighly mutagenic Pol V is produced (4). If the burden of DNAdamage is high, prolonging the SOS response, the cell eventuallyup-regulates production of Pol V to effectuate a salvage programto deal with unrepaired lesions.

SOS Response Can Be Activated by ssDNA Gaps Without Fork Break-down. It is widely accepted that the presence of ssDNA is thetrigger for activation of the SOS response by RecA (31).Research showing that replication is necessary for SOS inductionhas been interpreted to suggest that one fork must stall or breakdown, uncoupling helicase unwinding from DNA synthesis, togenerate ssDNA (19). However, cellular studies have shown thatDNA damage leads to single-strand gaps in both leading andlagging strands, and therefore replication forks skip over lesions,leaving ssDNA gaps behind them (32–35). These gaps may thenserve as substrate for RecA binding and consequent induction ofthe SOS response.

Gaps in the lagging strand are made possible by the numerouspriming events that occur during discontinuous synthesis. Spe-cifically, when a polymerase extending an Okazaki fragmentstalls at a lesion, it can transfer from the incomplete fragment toa new RNA primer, leaving behind a ssDNA gap. Recent studiesdemonstrate repriming of DNA synthesis on the leading strandas well, and therefore the same process can explain leadingstrand gaps (32, 36). Formation of ssDNA gaps behind thereplication fork offers an alternative means to generate ssDNAfor SOS induction that does not require fork stalling or break-down. The ability to skip over damage implies that replicationforks do not necessarily stall at every leading-strand lesion, butif Pol III were to continue rapid synthesis in the presence ofsignificant DNA damage, encounters with additional lesions,and the risk of stalling or collapse would increase. By slowing thefork, TLS polymerases diminish this risk while also assuring thatthe fork does not break down. It is important to note that whenthe lesion is a nick, replication forks will collapse, explaining theneed for primosomal replication restart proteins.

How Are Mutations Prevented If Error-Prone TLS Replisomes AreLong-Lived? Pol II and Pol IV replicate DNA with lower fidelitythan Pol III, and therefore an obvious concern about alternativeTLS replisomes is the increased risk of introducing heritablemutations, undermining rather than enhancing genome integ-rity. However, the cell contains several safeguards that prevent

Fig. 6. TLS polymerases function with DnaB and � clamps. (Left) Scheme ofPol III-based replisome. The �-subunit of Pol III binds the DnaB helicase andenables rapid unwinding. Two �-subunits are contained within a clamp loaderassembly (the clamp loader is not illustrated) and thereby dimerize 2 Pol IIImolecules for coupled leading- and lagging-strand synthesis. (Right) TLSpolymerases trade places with one another on the �-clamp with Pol III to forma TLS replisome. The TLS polymerases function with DnaB helicase, whichslows to match their slow rate of synthesis. Leading and lagging strands maybe uncoupled in the TLS replisome.

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production of heritable mutations under these circumstances.First, errors produced by a TLS enzyme operating on undam-aged DNA result in simple mismatches that can be corrected bythe postreplication mismatch repair system. Second, TLS DNApolymerases are very slow, thereby ensuring that mismatchescaused by an inherently low-fidelity polymerase are not pro-duced often. For example Pol IV, which makes a mistakeapproximately every 1,000 nt, moving at a speed of 1 ntd/s willmake only about 4 mismatches in 1 h. Correcting these errors isa relatively small task for the mismatch repair system. Third, PolII is a high-fidelity enzyme because it contains a proofreadingexonuclease. Finally, Pol IV is held in a high-fidelity state in theearly phase of the SOS response by interaction with full-lengthUmuD2 and RecA (6, 37). When the SOS response is prolonged,indicating the presence of significant or unrepairable damage,UmuD2 is cleaved to a form that no longer modulates the fidelityof Pol IV interacts with UmuC to form the low-fidelity Pol VTLS polymerase, thereby defining the later, mutagenic phase ofthe SOS response. Regulation of the fidelity of Pol IV by UmuDalso explains why overexpression of Pol IV alone was found tobe mutagenic whereas co-overexpression of Pol IV along with anuncleavable form of UmuD, UmuD(S60A), was not (37, 38).One may presume that during a catastrophic and prolonged SOSresponse, bypass of lesions by Pol V is of utmost importance,even at the cost of forming heritable mutations.

Experimental ProceduresMaterials. Subunits of Pol III holoenzyme (�, �, �, �, �, �, ��, �, , and �) werepurified as described (20). DnaB, DnaG, and SSB were purified as described(39). Pol III* (all holoenzyme subunits except �) was reconstituted as described(14). The C-terminal 5-residue deletion of Pol II (polB �779–783) and 6-residuedeletion of Pol IV (dinB �346–351) containing a His6 tag were cloned into thepET16 expression vector and purified by using Ni2� affinity chromatography;their activity was nearly that of WT Pol II and Pol IV (�70%). The tailed formII duplex minicircle DNA was as described (20). The BW27786 strain was a

generous gift from Jay Keasling (University of California, Berkeley). Buffer Ais 20 mM Tris�Cl (pH 7.5), 0.1 mM EDTA, 5 mM DTT, 5% glycerol, 40 g/ml BSA,0.5 mM ATP, and 8 mM MgCl2.

Rolling Circle Replication Reactions. DnaB was assembled on the minicircleDNA by incubating 100 fmol of DNA with 4 pmol of DnaB (as hexamer) in 15L of buffer A for 30 s at 37 °C, at which time Pol III* (100 fmol), � (350 fmol),and 60 M each of dGTP and dCTP were added for 5 min. DNA synthesis wasinitiated upon adding 1 g of SSB, 50 M of the 4 rNTPs, 60 M DnaG primase,60 M dATP, and 20 M dTTP (leading strand) or 60 M dTTP and 20 M dATP(lagging strand) in 25 L. DNA synthesis was monitored by using [�32P]dTTP forthe leading strand and [�32P] dATP for the lagging strand (3,000–5,000cpm/pmol). Pol II or Pol IV were added as indicated in the figure legends.Reactions lacking Pol III* contained 5 pmol of Pol IV (or Pol II), 50 fmol of�-complex, and 350 fmol of � (Fig. 1). All reactions were quenched upon theaddition of 0.5% SDS and 20 mM EDTA, and DNA synthesis was quantitated asdescribed (40). Products were analyzed on 0.6% alkaline agarose gels.

Measurement of DNA Synthesis After Protein Overexpression. The protocolused [3H]thymidine to monitor DNA synthesis after overexpression of differ-ent DNA polymerases as described (41). Dose-dependent expression of Pol II orPol IV was obtained by using the araC-pBAD system (Invitrogen). The plasmidwas transformed into E. coli strain BW27786, which overexpresses the arabi-nose permease, araE, allowing consistent and highly regulated expression ofpBAD in a population of cells (42). A single colony was inoculated in 5 mL ofLB, overnight cultures were diluted 1:100 in 100 mL of LB, then grown to OD600

� 0.1. 45 min after adding arabinose (0.1% for Pol II and 0.01% for Pol IV), and0.5-ml aliquots were removed and added to a tube containing pulse-labelsolution {30 L of [methyl-3H] thymidine (1mCi/mL) in 2.97 mL of LB}. Afterexactly 3 min at 37 °C, ice-cold 5% trichloroacetic acid was added to lyse cellsand precipitate DNA. Pol II and Pol IV were quantitated by Western blotanalysis. Control samples (�Ara) were from parallel cultures not treated witharabinose.

ACKNOWLEDGMENTS. We thank Jay Keasling for the AraE overexpressionstrain. This work was supported by National Institutes of Health GrantsGM38839 (to M.O.), AI065508 (to M.O.), R37GM21422 (to M.F.G.), and ESO12259 (to M.F.G.).

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