Chiara 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.
INAUGURAL ARTICLE SEE COMMENTARY
Translesion DNA polymerases remodel the replisome and alter the speed of the replicative helicase
All cells contain specialized translesion DNA polymerases that replicate past sites of DNA damage. We find that Escherichia coli translesion 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 freely exchange with the polymerase III (Pol III) replicase on the -clamp and function with DnaB helicase to form alternative replisomes, even before Pol III stalls at a lesion. DNA damage-induced levels of Pol II and Pol IV dominate the clamp, slowing the helicase and stably maintaining the architecture of the replication machinery while keeping the fork moving. We propose that these dynamic actions provide additional time for normal excision repair of lesions before the replication fork reaches them and also enable the appropriate translesion polymerase to sample each lesion as it is encountered. DNA repair 兩 DNA replication 兩 replication fork 兩 replisome sliding clamp
C
hromosomes are duplicated by replisome machines containing helicase, primase, and DNA polymerase activites (1). Replicative DNA polymerases are tethered to DNA by ringshaped sliding clamp proteins. The Escherichia coli polymerase III (Pol III) holoenzyme replicase contains 10 different subunits consisting of a clamp loader that binds 2 molecules of Pol III, each tethered to DNA by the -sliding clamp for processive leading and lagging strand synthesis (2). Pol III also binds tightly to the hexameric DnaB helicase that encircles the lagging strand template. The Pol III-to-DnaB interaction is mediated by the -subunit within Pol III (3). This tight connection couples Pol III motion to DnaB unwinding and increases the rate of unwinding by DnaB helicase from a basal level of 35 bp/s to a rate in excess of 500 bp/s (3). These tight connections of Pol III to DnaB and the -clamp provide the replisome with sufficient stability, in principle, to replicate the entire genome. In practice, however, replication of long chromosomal DNA is an uneven path with a variety of obstacles along the way, including protein blocks and damaged templates. In the presence of high levels of DNA damage, ⬎40 gene products are induced by the SOS response that act to restore genomic integrity and assure cell survival (4). Among the SOS-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 synthesis across template lesions (5). Pols IV and V are members of the Y family of error-prone DNA polymerases; they lack a proofreading 3⬘–5⬘ exonuclease (6, 7). Pol II is a B-family polymerase; it has high fidelity and contains proofreading activity (8). Pols II and IV are present during normal cell growth and may be involved in repairing low levels of DNA damage that occur routinely in the cell (9–11). Pol V, however, is closely associated with mutagenesis and is not detectable in cells under normal conditions (7, 12, 13). Pol II and Pol IV are among the first genes that are upregulated in the SOS response, whereas the levels of Pol V are www.pnas.org兾cgi兾doi兾10.1073兾pnas.0901403106
very slow to rise, peaking 45 min after SOS induction (4, 9, 11, 12). In vitro studies have shown that these DNA polymerases function with the -clamp for lesion bypass and trade places on  with a stalled Poll III replicase, as originally demonstrated in bacteriophage T4 (14–17). Once the lesion is bypassed, the high-fidelity Pol III can switch back on the -clamp to reestablish the fast moving E. coli replisome (650 nt per s) (14, 17). In this view of translesion synthesis, the TLS polymerase only occupies the -clamp during the short time interval of lesion bypass. It seems unlikely that TLS polymerases would be able to replace Pol III in the context of a moving replisome, because TLS polymerases are very slow and have low fidelity. These characteristics are inconsistent with the rapid speed and high fidelity required for chromosome replication. It is possible that TLS polymerases are excluded from replisomes by the tight connection between Pol III and the DnaB helicase via the -subunit. Additional insurance against TLS polymerase entry into the replisome is the known action of DnaB helicase when it becomes uncoupled from Pol III, whereupon it continues for only ⬇1 kb before DnaB stops unwinding (18–20). Presumably, DnaB dissociates from DNA when it is uncoupled from leadingstrand Pol III action. Once DnaB dissociates, it cannot get back onto DNA without specialized helicase-loading factors, and replication fork progression is halted. Therefore, even if a TLS polymerase enters the replisome, the subsequent loss of Pol III interaction with DnaB may result in DnaB dissociation, preventing further progression of the fork. Fork breakdown upon takeover by Pol IV is supported by recent studies that show that overexpression of Pol IV in the absence of DNA damage inhibits cell growth in Bacillus subtilis and E. coli (21, 22). The authors of the E. coli study (21) note that induction 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 clamp binding residues of Pol IV, suggesting that Pol IV blocks replication by a process distinct from TLS polymerase switching on a clamp. However, Pol IV was overexpressed to abnormally high levels in that study. Thus, the mechanism of the replication block by Pol IV was suggested to be separate from the physiological action of the enzyme. Lower levels of Pol IV expression appeared to decrease replication without cell killing, suggesting Pol IV may slow a replication fork, but this hypothesis could not be 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:
[email protected].
This article contains supporting information online at www.pnas.org/cgi/content/full/ 0901403106/DCSupplemental.
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Contributed by Mike O’Donnell, February 11, 2009 (sent for review January 22, 2009)
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.
In the current study we examine the effects of normal and induced levels of Pol II and Pol IV on moving replisomes reconstituted in vitro. We find that these TLS polymerases can gain access to the moving Pol III replisome despite the strong Pol III––DnaB connection. Surprisingly, the fork does not break down. Instead the TLS polymerases function with DnaB to form replisomes that move more slowly than the intrinsic rate of DnaB, with the rate of helicase unwinding adjusting to the speed of the polymerase at the fork. At the levels of Pol II and Pol IV present in undamaged cells, the Pol III replisome is unaffected, whereas at levels of Pol II and Pol IV that correspond to their concentrations in DNA damage-induced cells, the TLS polymerases dominate the replication fork and slow it down. Expression of Pol II and Pol IV in vivo in the absence of DNA damage also slows DNA replication and depends on TLS polymerase interaction with the -clamp. Slow replication fork advance in the context of DNA damage makes biological sense as it buys time for normal DNA repair, decreasing collision events of the fork with DNA lesions. Results Pol II and Pol IV Form Alternative Replisomes with the -Clamp and DnaB Helicase. To investigate possible alternative roles for Pol II
and Pol IV beyond translesion synthesis, we examined their ability to form a functional replisome with the -clamp and DnaB helicase in the absence of Pol III. For these studies, we used a minicircle replication fork substrate consisting of a synthetic 100-mer circular duplex with a 5⬘ T40 ssDNA tail (20, 23). Each strand of the synthetic minicircle substrate contains only 3 nt: dG, dC, dA on the leading strand template, and dG, dC, dT on the lagging stand template. Hence, leading and lagging strands can be specifically labeled in separate reactions using either [␣32P] dTTP or [␣32P] dATP, respectively. The replication machinery at the fork was assembled by loading DnaB on the 5⬘ dT40 tail of the minicircle DNA in the 6032 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0901403106
absence of ssDNA-binding protein (SSB), then the -clamp was loaded onto DNA by using the clamp loader followed by the addition of Pol II or Pol IV in the presence of only dCTP and dGTP to prevent fork movement. After 5 min, replication was initiated upon adding dATP, dTTP, SSB, DnaG primase, and the 4 rNTPs (see Fig. 1A). In these assays, dissociation of DnaB from DNA will halt the fork because SSB is present and the assay does not contain DnaB-reloading factors. Replication fork progression is followed by analysis of timed aliquots in an alkaline agarose gel. The results are compared with similar reactions performed in the absence of DnaB helicase or the absence of the clamp and clamp loader. Surprisingly, the analysis shows that Pol II and Pol IV function with DnaB and the -clamp, because the presence of both of these factors is required to synthesize long DNA chains (Fig. 1 B and C, lanes 1–6). DNA synthesis continues for at least 20 min, indicating that the DnaB helicase acts processively and is maintained the entire time. The rates of Pol II and Pol IV fork progression are ⬇10 nucleotides (ntd)/s and 1 ntd/s, respectively (Fig. S1), significantly slower than Pol III replisomes under the same conditions and consistent with the slower synthetic rates of Pol II and Pol IV compared with Pol III (24, 25). The ability of Pol II and Pol IV to function with DnaB in the absence of Pol III indicates that replisomes containing these TLS polymerases remain intact rather than undergo replication fork breakdown. These results also suggest that the speed of a replicative helicase can be limited by the rate of synthesis of the polymerase traveling along behind it. Lagging strand products are also observed by using [␣32P] dATP as the radiolabel as shown in Fig. S2. We presume that leading and lagging strand synthesis is uncoupled under these conditions, as further described in Discussion. Both Pol II and Pol IV are present in normal cells and are greatly up-regulated during the SOS response. As shown in Fig. 1D, the rate of DNA synthesis in the presence of Pol II and Pol IV (Center and Right) was slower than that of Pol II alone (Left), Indiani et al.
suggesting that both polymerases have access to the fork and alternate in taking control of the growing DNA ends in a dynamic fashion. If the 2 polymerases did not repeatedly trade places at the fork, 2 distinct products would be observed that correspond to the rates of Pol II- and Pol IV-driven replication forks. We confirmed that Pol II acts in a distributive manner at the replication fork by showing that the speed of Pol II replisomes 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 of the Pol II and Pol IV replisomes and presumably stimulates synthesis by unwinding DNA. However, DnaB is not known to function with other DNA polymerases, nor is DnaB known to unwind DNA as slowly as observed in these alternative replisomes. Hence, it remained possible that DnaB helicase is acting in some other capacity to stimulate synthesis on the minicircle replication fork substrate. If DnaB is truly functional and encircles DNA in the alternative replisomes, then Pol III should be capable of displacing the TLS polymerase with resumption of the rapid unwinding characteristic of the coupled DnaB–Pol III replisome. To examine this issue and provide further evidence that replisomes containing Pol II or Pol IV are fully functional and have not broken down, we performed a series of pulse–chase experiments by adding Pol III to a prelabeled replication fork containing a moving Pol II or Pol IV (see Fig. 2A). Pol II or Pol IV was first assembled with DnaB, the sliding clamp, and clamp loader in a 5-min preincubation, and then DNA synthesis was initiated for 7 min (Pol II) or 15 min (Pol IV) in the presence of [␣32P] dTTP (pulse). After this labeling reaction, a large excess Indiani et al.
INAUGURAL ARTICLE SEE COMMENTARY
Pol II and Pol IV Recruit the Clamp and Helicase from a Moving Pol III Replisome. Our previous studies with singly-primed ssDNA cir-
cles showed that when Pol III holoenzyme was stalled at a primed site by nucleotide omission Pol IV takes control of the primer terminus even at low concentrations of Pol IV, but when Pol III is engaged in active DNA synthesis, much higher levels of Pol IV are required to take over the primed site (14). However, at a replication fork the Pol III holoenzyme has several additional connections that may anchor it to DNA and block entry of other DNA polymerases. Specifically, at a replication fork Pol III connects to 2 -clamps on both the leading and lagging strands through its twin polymerase structure. Furthermore, Pol III holoenzyme also connects to the DnaB helicase via the -subunit (26). These Pol III-specific connections may prevent takeover of a Pol III replisome by Pol II or Pol IV. Based on the results of Figs. 1 and 2, one would predict that if Pol II or Pol IV can recruit the -clamp from a Pol III replisome, the result would be a functional, but slow, alternative replisome. To examine the ability of TLS polymerases to take over a moving replication fork, increasing concentrations of Pol II and Pol IV were added to a moving Pol III-based replisome in the minicircle replication fork assay along with [␣32P]dTTP (leading strand) or [␣32P]dATP (lagging strand), as illustrated in Fig. 3A. Timed aliquots were collected and analyzed in an alkaline agarose gel. The first 4 lanes in Fig. 3B show the long leading-strand products typically formed by the fast-moving Pol III replisome, as reported (20). Reactions were then analyzed in the presence of increasing amounts of Pol II (Fig. 3B Left, lanes 5–20) or Pol IV (Fig. 3B Right, lanes 5–20). The results indicate that both Pol II and Pol IV interfere with the accumulation of long leading-strand products generated by Pol III in a dosedependent manner. Inspection of the gel clearly shows that Pol IV is more efficient at inhibiting the replisome than Pol II; rolling circle replication was reduced ⬇50% upon addition of 0.5 pmol of Pol IV (Fig. 3B Right, lanes 9–12), whereas 2 pmol of Pol II was required for a 40% decrease of DNA synthesis (Fig. 3B Left, lanes 13–16). Lagging-strand replication was reduced to the same extent as leading-strand synthesis (Fig. S4). All 5 E. coli polymerases function with the clamp and bind  PNAS 兩 April 14, 2009 兩 vol. 106 兩 no. 15 兩 6033
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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. 1 A. After a 7-min pulse (Pol II) or 15-min pulse (Pol IV), a 100-fold excess of unlabeled dNTPs (chase) was added 10 s before Pol III (1 pmol), and reactions were quenched at different times after the addition of Pol III. Radiolabeled DNA synthesized by the TLS polymerase during the pulse period is shown in red, and DNA synthesized by Pol III during the chase period is in blue. (B) DNA synthesis by Pol II (Left) or Pol IV (Right) without Pol III and cold dNTPs (lanes 1, 5, and 9), with cold dNTPs (lanes 2, 6, and 10), and with Pol III and cold dNTPs (lanes 3, 7, and 11). Control experiments where Pol III was added to an unoccupied fork after addition of cold dNTPs are shown in lanes 4, 8, and 12.
of unlabeled dNTPs (chase) was added to prevent incorporation of additional [␣32P] dTTP. Then, Pol III holoenzyme lacking the -clamp (referred to as Pol III*) was added. If Pol III* takes control of the replication fork, the radiolabel incorporated by Pol II or Pol IV during the pulse period will quickly shift to a higher molecular mass during the chase period because of the rapid rate of synthesis by Pol III*-. As shown in Fig. 2B, Pol II (Left) synthesized ⬇3 kb during the 7-min pulse of [␣32P] dTTP, and Pol 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 large excess of cold dNTPs had no effect on the rate of synthesis by Pol II or Pol IV in the absence of added Pol III* (Fig. 2B, lanes 2, 6, and 10), but when Pol III* was added immediately after the chase, the pulse-labeled band shifted dramatically higher, in a time-dependent manner (Fig. 2B, lanes 3, 7, and 11). As a control for the effectiveness of the chase, Pol III* was added to replication forks assembled without Pol II or Pol IV and showed no detectable incorporation of [␣32P] dTTP (Fig. 2B, lanes 4, 8, and 12). This pulse–chase experiment confirms that the DnaB helicase is fully functional within the alternative Pol II and Pol IV replisomes and that the basic architecture of the machinery at the replication fork is maintained by the specialized DNA polymerases despite their slow speed compared with Pol III*-. Hence, DnaB is stable on DNA in a functional state, and its rate of DNA unwinding is determined by the speed of the DNA polymerase that functions with it. We also conclude that Pol III* takeover of Pol II and Pol IV replisomes is rapid and efficient.
Fig. 4. Pol II and Pol IV slow down the replication fork. (A) The Pol III-based replisome was assembled as described in Fig. 3A. DNA synthesized by Pol III is in blue, and DNA synthesized by the TLS polymerase is in red. (B) After 25 s (0 time point, lanes 1 and 9) a high concentration of Pol II (27 pmol; Left) or Pol IV (35 pmol; Right) was added, and aliquots were collected (lanes 2– 8). Time courses of Pol III alone (lanes 9 –16) are shown for comparison. The dotted red line highlights the difference in DNA chain length at 120 s with and without added Pol II. Fig. 3. Pol II and Pol IV takeover of Pol III-based replisomes. (A) The replisome is assembled on DNA as in Fig. 1, except Pol III* is used instead of Pol II or Pol IV. After 10 s, different amounts of TLS polymerase (pink) are added together with [␣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 the presence of 0 pmol (lanes 1– 4), 0.1 pmol (lanes 5– 8), 0.5 pmol (lanes 9 –12), 2 pmol (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 III on a minicircle substrate. Leading-strand synthesis is shown (Pol III only is in lanes 1– 6).
fork by the -subunit of the clamp loader, and this connection is essential for rapid, processive rolling circle replication by Pol III*. Because Pol III is poorly processive in the absence of , we were not able to determine whether plays a role in the takeover of a Pol III replisome by Pol II or Pol IV. We did, however, determine that is not required for rolling circle replication by Pol II (Fig. S5), suggesting that the role played by in stimulating DnaB-dependent replication might be unique to Pol III, perhaps by making Pol III more processive in the context of the replication fork rather than by directly stimulating unwinding by DnaB.
through a conserved consensus sequence (27). Pol II and Pol IV bind to  via the C-terminal 5 or 6 amino acid residues, respectively (24, 28). To determine whether Pol II and Pol IV function with  in takeover of Pol III-based replisomes, we expressed and purified mutant versions of Pol II and Pol IV that lack their respective C-terminal -binding residues and tested them in the minicircle replication assay outlined in Fig. 3A. For these experiments, 5 pmol of either WT or mutant Pol II or Pol IV was added 10 s after initiation of replication by Pol III. As expected for -dependent function, the C-terminal mutant Pol II and Pol IV were no longer capable of inhibiting Pol III-based replisomes in the minicircle replication assay (compare WT lanes 7–12 vs. ⌬C-term lanes 13–18 for Pol II, Fig. 3C Left, and Pol IV, Fig. 3C Right). These results support the conclusion that Pol II and Pol IV take control of the replisome from Pol III by competitive 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-filling assays that do not require . Pol III is connected to the DnaB helicase at the replication
Pol II and Pol IV Remodel the Pol III-Based Replisome and Slow Replication Fork Advance. The results of Fig. 3 indicate that when
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either Pol II or Pol IV is added to a Pol III-based replisome, the replisome is remodeled to include the TLS polymerase, leading to a slower moving fork that does not break down but remains intact. Nonetheless, it remains possible that Pol II and Pol IV simply inhibit the replication reaction without taking control of the replication fork or stop the fork and cause it to break down. Hence, we designed the experiments illustrated in the scheme of Fig. 4A to determine whether the replication fork continues to slowly advance after the addition of Pol II or Pol IV. Because Pol III-based replisomes quickly produce replication products that approach the resolution limit of alkaline gels, we examined very short time points after addition of the TLS polymerase. In the case of Pol II, it is clear that the fork does not break down; fork progression continues but at a slower rate (Fig. 4B Left). Pol IV appears to stop the replication fork (Fig. 4B Right), but considering that the Pol IV fork only moves 1 ntd/s it would only extend DNA by 120 nt at the final time point of this experiment. This short extension cannot be detected considering that the Pol III Indiani et al.
replisome has already moved nearly 10 kb by the time Pol IV is added. Based on the results of Fig. 1C, showing that Pol IV functions with DnaB, and Fig. 2B, showing that Pol IV replisomes are competent for resumption of synthesis by Pol III*, we presume that takeover of the fork by Pol IV causes the fork to slow dramatically rather than break down. Induction of Pol II and Pol IV in the Cell Reduces Chromosome Replication in the Absence of DNA Damage. The in vitro reactions
of 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 the replisome and slow down the rate of chromosome replication in vivo. To test this prediction, the genes encoding Pol II and Pol IV were cloned into a plasmid under control of the inducible arabinose promoter, and the incorporation of [3H] thymidine during replication was measured before and after induction (see Fig. 5A). The amounts of arabinose used for induction yielded 1,200 molecules of Pol II per cell and 2,500 molecules of Pol IV per cell, within 2- to 3-fold the levels present in the cell during the SOS response (9, 10). The results of Fig. 5B indicate that induction of Pol II is accompanied by a 55% decrease in DNA replication, and induction of Pol IV decreases DNA synthesis by ⬇99% (Fig. 5C). The more severe decrease in DNA synthesis upon overexpression of Pol IV compared with Pol II correlates well with the results of the in vitro assays of Fig. 3. Control experiments using the same plasmid, but without the Pol II or Pol IV genes, showed that the vector does not interfere with chromosomal replication. Indiani et al.
Discussion This article demonstrates that E. coli translesion DNA polymerases Pol II and Pol IV form alternative replisomes with DnaB helicase (Fig. 1) and maintain the architecture of the replication machinery for eventual resumption of synthesis by Pol III (Fig. 2). Whether overexpressed in the cell or studied in vitro, high levels of Pol II and Pol IV clearly interfere with ongoing processive synthesis by Pol III (Figs. 3 and 5), by slowing rather than stopping the replisome (Fig. 4), and this inhibition of Pol III replication depends on the interaction of Pol II and Pol IV 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 Pol II and Pol IV recruit both the -clamp and DnaB helicase from Pol III at a moving replication fork during the SOS response. Surprisingly, these TLS polymerases modulate the rate of the DnaB helicase and slow the replication fork without causing it to break down. DnaB as a Regulated Motor. DnaB is not known to function with
other polymerases besides Pol III, yet Pol II and Pol IV slow the rate of DnaB unwinding, suggesting they may connect to DnaB and regulate helicase speed. A reinterpretation of previous studies suggests another explanation. Specifically, the rate of DnaB unwinding may simply be determined by the rate of any DNA polymerase that acts on the unwound ssDNA product as illustrated in bacteriophage T4 and T7 systems (43, 44). The 35 bp/s rate of DnaB unwinding observed in an earlier study was determined by monitoring chain extension by Pol III in the absence of the -subunit (3). Because , which binds DnaB, was not present it was thought that the observed rate of unwinding was the intrinsic rate of DnaB. Instead, the observed unwinding rate may represent the rate of DnaB unwinding when functioning with Pol III core. The same study also observed a 35 bp/s rate by the primosome, which contains DnaB and other proteins, including DnA helicase (3). The current study shows that Pol II PNAS 兩 April 14, 2009 兩 vol. 106 兩 no. 15 兩 6035
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Fig. 5. Expression of Pol II and Pol IV in vivo reduces chromosomal DNA synthesis. The rate of DNA synthesis is expressed as incorporation of [3H]thymidine over a 3-min interval starting 45 min after overexpression of the TLS polymerase by the addition of arabinose. (A) Scheme of the experiment as detailed in Experimental Procedures. (B) Arabinose-induced expression of Pol II WT. (C) Pol IV WT. (D) Pol II ⌬C-term. (E) Pol IV ⌬C-term. Pol II ⌬C-term and Pol IV ⌬C-term are impaired in their ability to bind the clamp. For B–E, 3H incorporation was adjusted to account for different cell densities of uninduced and induced cultures.
To test whether the replication inhibition observed upon expression of Pol II and Pol IV in vivo depends on interaction of the 2 specialized polymerases with the -clamp, genes encoding Pol II and Pol IV mutants that lack the C-terminal -interacting residues were cloned into the arabinose inducible plasmid and tested for [3H] thymidine incorporation in the presence and absence of arabinose. The mutant polymerases were found to have no measurable effect on DNA synthesis. The results in Fig. 5 D and E indicate that Pol II and Pol IV interact with  to reduce intracellular replication, consistent with the in vitro results of Fig. 3C. Although we were unable to distinguish whether DNA synthesis in vivo was slowed or stopped by overexpression of Pol II and Pol IV, the results of Fig. 5 are remarkably consistent with the in vitro experiments of Fig. 3, suggesting that the reconstituted rolling circle replication system can be mimicked by Pol II and Pol IV overexpression in living cells. Despite the good agreement of the in vivo and in vitro data, we cannot eliminate the possibility that differences in sulA inhibition of cell growth could reflect a differential induction of the SOS response when WT Pol II or Pol IV are overexpressed, and thereby might account, at least in part, for the differences in DNA synthesis (Fig. 5 B and C). It is interesting to note that UV irradiation of Pol II mutant cells delays recovery of replication restart (29), which may be caused, in part, by more efficient takeover by Pol IV in the absence of Pol II (i.e., Pol IV is the more potent of the 2 polymerases in slowing the fork). However, a subsequent study did not observe a defect in the rate of recovery after UV irradiation of either Pol II or Pol IV mutant cells (30), suggesting that the rate of recovery depends on the kinetics of the repair processes themselves and not on whether replication proceeds slowly while repair is occurring behind the fork.
Fig. 6. TLS polymerases function with DnaB and  clamps. (Left) Scheme of Pol III-based replisome. The -subunit of Pol III binds the DnaB helicase and enables rapid unwinding. Two -subunits are contained within a clamp loader assembly (the clamp loader is not illustrated) and thereby dimerize 2 Pol III molecules for coupled leading- and lagging-strand synthesis. (Right) TLS polymerases trade places with one another on the -clamp with Pol III to form a TLS replisome. The TLS polymerases function with DnaB helicase, which slows to match their slow rate of synthesis. Leading and lagging strands may be uncoupled in the TLS replisome.
and Pol IV, which are slower than Pol III, function with DnaB to move replication forks at rates of 10 and 1 bp/s, respectively. If the intrinsic rate of DnaB unwinding is truly 35 bp/s, the current study indicates that Pol II and Pol IV slow DnaB. The highest rate of DnaB unwinding is observed by using Pol III holoenzyme containing (⬎500 bp/s). In light of these 4 different rates of unwinding, ranging ⬎500-fold, we propose that the DnaB unwinding rate is coupled to DNA synthetic rate, even in the absence of a direct coupling factor 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 ensure that unwinding is tightly coordinated with DNA synthesis, a favorable property for replisome action. Otherwise, ssDNA will be generated that, besides being vulnerable to nucleases and strand 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 polymerases has been considered an ephemeral phenomenon, where a stalled replicative polymerase briefly yields to the TLS polymerase only for bypass of the lesion, and Pol III immediately retakes control of the growing DNA end to resume processive replication. The results shown here compel a reconsideration of this notion, because high levels of Pol II and Pol IV, as found in the early stages of the SOS response, clearly take over replication by Pol III to form slower replisomes that function not only with the clamp but also with the replicative DnaB helicase. Unlike Pol III replicase that contains 2 Pol III cores connected to a clamp loader, Pol II and Pol IV are not known to dimerize or connect to other proteins besides . Hence, we presume that leading- and lagging-strand synthesis in TLS replisomes are constitutively uncoupled, with no need to hypothesize a DNA trombone loop (illustrated in Fig. 6). We propose that -clamps are assembled on lagging strand RNA primers by the ␥-complex, and perhaps it may be the reason for production of both ␥ and in cells. Nevertheless, it remains possible that TLS polymerases dimerize or connect to other proteins in the context of a replication fork. The Pol II and Pol IV replisomes are highly stable and therefore are probably the active form during the entire SOS response. These replisomes are also highly dynamic. We dem6036 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0901403106
onstrate here that all 3 DNA polymerases (Pols II, III, and IV) freely exchange with one another while preserving the DNAbound helicase and sliding clamps. We presume the average residence time of any particular polymerase at the fork is defined by the relative amounts of the different DNA polymerases in the cell, their processivity, and their affinity for the sliding clamp and helicase. Likewise, the speed of the replication fork will be a function of the residence time of each DNA polymerase and its intrinsic rate of DNA polymerization. As the intracellular concentration of TLS polymerases decreases, the population time of Pol III at the replication fork will increase, with a corresponding increase in replication fork speed. What would be the purpose of establishing slower replication forks in the face of DNA damage? We propose an additional role for SOS-induced Pol II and Pol IV besides their ability to bypass lesions. Specifically that TLS polymerases slow the fork, which may give more time for repair of DNA damage by the excision repair system, which can only function on dsDNA before a replication fork collides with a lesion. Of course, sometimes the replisome will encounter a lesion, and TLS synthesis will bypass it, producing a mutation. Nevertheless, slower moving forks should lower the frequency of these events. Pol II and Pol IV are induced early in the SOS damage response and therefore replication fork control by these polymerases is expected to be specific to the first phase (⬍45 min) of SOS induction before the highly mutagenic Pol V is produced (4). If the burden of DNA damage is high, prolonging the SOS response, the cell eventually up-regulates production of Pol V to effectuate a salvage program to deal with unrepaired lesions. SOS Response Can Be Activated by ssDNA Gaps Without Fork Breakdown. It is widely accepted that the presence of ssDNA is the
trigger for activation of the SOS response by RecA (31). Research showing that replication is necessary for SOS induction has been interpreted to suggest that one fork must stall or break down, uncoupling helicase unwinding from DNA synthesis, to generate ssDNA (19). However, cellular studies have shown that DNA damage leads to single-strand gaps in both leading and lagging strands, and therefore replication forks skip over lesions, leaving ssDNA gaps behind them (32–35). These gaps may then serve as substrate for RecA binding and consequent induction of the SOS response. Gaps in the lagging strand are made possible by the numerous priming events that occur during discontinuous synthesis. Specifically, when a polymerase extending an Okazaki fragment stalls at a lesion, it can transfer from the incomplete fragment to a new RNA primer, leaving behind a ssDNA gap. Recent studies demonstrate repriming of DNA synthesis on the leading strand as well, and therefore the same process can explain leading strand gaps (32, 36). Formation of ssDNA gaps behind the replication fork offers an alternative means to generate ssDNA for SOS induction that does not require fork stalling or breakdown. The ability to skip over damage implies that replication forks do not necessarily stall at every leading-strand lesion, but if Pol III were to continue rapid synthesis in the presence of significant DNA damage, encounters with additional lesions, and the risk of stalling or collapse would increase. By slowing the fork, TLS polymerases diminish this risk while also assuring that the fork does not break down. It is important to note that when the lesion is a nick, replication forks will collapse, explaining the need for primosomal replication restart proteins. How Are Mutations Prevented If Error-Prone TLS Replisomes Are Long-Lived? Pol II and Pol IV replicate DNA with lower fidelity
than Pol III, and therefore an obvious concern about alternative TLS replisomes is the increased risk of introducing heritable mutations, undermining rather than enhancing genome integrity. However, the cell contains several safeguards that prevent Indiani et al.
Experimental Procedures Materials. Subunits of Pol III holoenzyme (␣, , , ␥, , ␦, ␦⬘, , , and ) were purified 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-residue deletion of Pol IV (dinB ⌬346 –351) containing a His6 tag were cloned into the pET16 expression vector and purified by using Ni2⫹ affinity chromatography; their activity was nearly that of WT Pol II and Pol IV (⬎70%). The tailed form II duplex minicircle DNA was as described (20). The BW27786 strain was a
1. Kornberg A, Baker TA (1992) DNA replication. DNA Replication (Freeman Press, New York), 2nd ed, p 931. 2. Johnson A, O’Donnell M (2005) Cellular DNA replicases: Components and dynamics at the replication fork. Annu Rev Biochem 74:283–315. 3. Kim S, Dallmann HG, McHenry CS, Marians KJ (1996) Coupling of a replicative polymerase and helicase: A tau–DnaB interaction mediates rapid replication fork movement. Cell 84:643– 650. 4. Courcelle J, Khodursky A, Peter B, Brown PO, Hanawalt PC (2001) Comparative gene expression profiles following UV exposure in wild-type and SOS-deficient Escherichia coli. Genetics 158:41– 64. 5. Napolitano R, Janel-Bintz R, Wagner J, Fuchs RP (2000) All three SOS-inducible DNA polymerases (Pol II, Pol IV, and Pol V) are involved in induced mutagenesis. EMBO J 19:6259 – 6265. 6. Kobayashi S, Valentine MR, Pham P, O’Donnell M, Goodman MF (2002) Fidelity of Escherichia coli DNA polymerase IV. Preferential generation of small deletion mutations by dNTP-stabilized misalignment. J Biol Chem 277:34198 –34207. 7. Tang M, et al. (2000) Roles of E. coli DNA polymerases IV and V in lesion-targeted and untargeted SOS mutagenesis. Nature 404:1014 –1018. 8. Cai H, Yu H, McEntee K, Kunkel TA, Goodman MF (1995) Purification and properties of wild-type and exonuclease-deficient DNA polymerase II from Escherichia coli. J Biol Chem 270:15327–15335. 9. Bonner CA, Hays S, McEntee K, Goodman MF (1990) DNA polymerase II is encoded by the DNA damage-inducible dinA gene of Escherichia coli. Proc Natl Acad Sci USA 87:7663–7667. 10. Kim SR, Matsui K, Yamada M, Gruz P, Nohmi T (2001) Roles of chromosomal and episomal dinB genes encoding DNA pol IV in targeted and untargeted mutagenesis in Escherichia coli. Mol Genet Genomics 266:207–215. 11. Qiu Z, Goodman MF (1997) The Escherichia coli polB locus is identical to dinA, the structural gene for DNA polymerase II. Characterization of Pol II purified from a polB mutant. J Biol Chem 272:8611– 8617. 12. Woodgate R, Ennis DG (1991) Levels of chromosomally encoded Umu proteins and requirements for in vivo UmuD cleavage. Mol Gen Genet 229:10 –16. 13. Woodgate R, Levine AS (1996) Damage inducible mutagenesis: Recent insights into the activities of the Umu family of mutagenesis proteins. Cancer Surv 28:117–140. 14. Indiani C, McInerney P, Georgescu R, Goodman MF, O’Donnell M (2005) A sliding-clamp toolbelt binds high- and low-fidelity DNA polymerases simultaneously. Mol Cell 19:805– 815. 15. Yang J, Zhuang Z, Roccasecca RM, Trakselis MA, Benkovic SJ (2004) The dynamic processivity of the T4 DNA polymerase during replication. Proc Natl Acad Sci USA 101:8289 – 8294.
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Rolling Circle Replication Reactions. DnaB was assembled on the minicircle DNA by incubating 100 fmol of DNA with 4 pmol of DnaB (as hexamer) in 15 L 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 was initiated 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 for the leading strand and [␣32P] dATP for the lagging strand (3,000 –5,000 cpm/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 the addition of 0.5% SDS and 20 mM EDTA, and DNA synthesis was quantitated as described (40). Products were analyzed on 0.6% alkaline agarose gels.
INAUGURAL ARTICLE SEE COMMENTARY
generous gift from Jay Keasling (University of California, Berkeley). Buffer A is 20 mM Tris䡠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.
Measurement of DNA Synthesis After Protein Overexpression. The protocol used [3H]thymidine to monitor DNA synthesis after overexpression of different DNA polymerases as described (41). Dose-dependent expression of Pol II or Pol IV was obtained by using the araC-pBAD system (Invitrogen). The plasmid was transformed into E. coli strain BW27786, which overexpresses the arabinose permease, araE, allowing consistent and highly regulated expression of pBAD in a population of cells (42). A single colony was inoculated in 5 mL of LB, 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), and 0.5-ml aliquots were removed and added to a tube containing pulse-label solution {30 L of [methyl-3H] thymidine (1mCi/mL) in 2.97 mL of LB}. After exactly 3 min at 37 °C, ice-cold 5% trichloroacetic acid was added to lyse cells and precipitate DNA. Pol II and Pol IV were quantitated by Western blot analysis. Control samples (⫺Ara) were from parallel cultures not treated with arabinose. ACKNOWLEDGMENTS. We thank Jay Keasling for the AraE overexpression strain. This work was supported by National Institutes of Health Grants GM38839 (to M.O.), AI065508 (to M.O.), R37GM21422 (to M.F.G.), and ESO 12259 (to M.F.G.).
16. Furukohri A, Goodman MF, Maki H (2008) A dynamic polymerase exchange with Escherichia coli DNA polymerase IV replacing DNA polymerase III on the sliding clamp. J Biol Chem 283:11260 –11269. 17. Fujii S, Fuchs RP (2004) Defining the position of the switches between replicative and bypass DNA polymerases. EMBO J 23:4342– 4352. 18. Higuchi K, et al. (2003) Fate of DNA replication fork encountering a single DNA lesion during oriC plasmid DNA replication in vitro. Genes Cells 8:437– 449. 19. Pages V, Fuchs RP (2003) Uncoupling of leading- and lagging-strand DNA replication during lesion bypass in vivo. Science 300:1300 –1303. 20. McInerney P, O’Donnell M (2004) Functional uncoupling of twin polymerases: Mechanism of polymerase dissociation from a lagging-strand block. J Biol Chem 279:21543–21551. 21. Uchida K, et al. (2008) Overproduction of Escherichia coli DNA polymerase DinB (Pol IV) inhibits replication fork progression and is lethal. Mol Microbiol 70:608 – 622. 22. Duigou S, Ehrlich SD, Noirot P, Noirot-Gros MF (2004) Distinctive genetic features exhibited by the Y-family DNA polymerases in Bacillus subtilis. Mol Microbiol 54:439 – 451. 23. Lee J, Chastain PD, 2nd, Kusakabe T, Griffith JD, Richardson CC (1998) Coordinated leading and lagging strand DNA synthesis on a minicircular template. Mol Cell 1:1001–1010. 24. Bonner CA, et al. (1992) Processive DNA synthesis by DNA polymerase II mediated by DNA polymerase III accessory proteins. J Biol Chem 267:11431–11438. 25. Wagner J, Fujii S, Gruz P, Nohmi T, Fuchs RP (2000) The -clamp targets DNA polymerase IV to DNA and strongly increases its processivity. EMBO Rep 1:484 – 488. 26. Gao D, McHenry CS (2001) tau binds and organizes Escherichia coli replication proteins through distinct domains. Domain IV, located within the unique C terminus of tau, binds the replication fork, helicase, DnaB. J Biol Chem 276:4441– 4446. 27. Dalrymple BP, Kongsuwan K, Wijffels G, Dixon NE, Jennings PA (2001) A universal protein–protein interaction motif in the eubacterial DNA replication and repair systems. Proc Natl Acad Sci USA 98:11627–11632. 28. Lenne-Samuel N, Wagner J, Etienne H, Fuchs RP (2002) The processivity factor  controls DNA polymerase IV traffic during spontaneous mutagenesis and translesion synthesis in vivo. EMBO Rep 3:45– 49. 29. Rangarajan S, Woodgate R, Goodman MF (1999) A phenotype for enigmatic DNA polymerase II: A pivotal role for pol II in replication restart in UV-irradiated Escherichia coli. Proc Natl Acad Sci USA 96:9224 –9229. 30. Courcelle CT, Belle JJ, Courcelle J (2005) Nucleotide excision repair or polymerase V-mediated lesion bypass can act to restore UV-arrested replication forks in Escherichia coli. J Bacteriol 187:6953– 6961. 31. Little JW, Mount DW, Yanisch-Perron CR (1981) Purified lexA protein is a repressor of the recA and lexA genes. Proc Natl Acad Sci USA 78:4199 – 4203. 32. Heller RC, Marians KJ (2006) Replication fork reactivation downstream of a blocked nascent leading strand. Nature 439:557–562.
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production of heritable mutations under these circumstances. First, errors produced by a TLS enzyme operating on undamaged DNA result in simple mismatches that can be corrected by the postreplication mismatch repair system. Second, TLS DNA polymerases are very slow, thereby ensuring that mismatches caused by an inherently low-fidelity polymerase are not produced often. For example Pol IV, which makes a mistake approximately every 1,000 nt, moving at a speed of 1 ntd/s will make only about 4 mismatches in 1 h. Correcting these errors is a relatively small task for the mismatch repair system. Third, Pol II is a high-fidelity enzyme because it contains a proofreading exonuclease. Finally, Pol IV is held in a high-fidelity state in the early phase of the SOS response by interaction with full-length UmuD2 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 fidelity of Pol IV interacts with UmuC to form the low-fidelity Pol V TLS polymerase, thereby defining the later, mutagenic phase of the SOS response. Regulation of the fidelity of Pol IV by UmuD also explains why overexpression of Pol IV alone was found to be mutagenic whereas co-overexpression of Pol IV along with an uncleavable form of UmuD, UmuD(S60A), was not (37, 38). One may presume that during a catastrophic and prolonged SOS response, bypass of lesions by Pol V is of utmost importance, even at the cost of forming heritable mutations.
33. Rupp WD, Howard-Flanders P (1968) Discontinuities in the DNA synthesized in an excision-defective strain of Escherichia coli following ultraviolet irradiation. J Mol Biol 31:291–304. 34. Sassanfar M, Roberts JW (1990) Nature of the SOS-inducing signal in Escherichia coli. The involvement of DNA replication. J Mol Biol 212:79 –96. 35. Amado L, Kuzminov A (2006) The replication intermediates in Escherichia coli are not the product of DNA processing or uracil excision. J Biol Chem 281:22635–22646. 36. Pomerantz RT, O’Donnell M (2008) The replisome uses mRNA as a primer after colliding with RNA polymerase. Nature 456:762–766. 37. Godoy VG, et al. (2007) UmuD and RecA directly modulate the mutagenic potential of the Y family DNA polymerase DinB. Mol Cell 28:1058 –1070. 38. Kim SR, et al. (1997) Multiple pathways for SOS-induced mutagenesis in Escherichia coli: An overexpression of dinB/dinP results in strongly enhancing mutagenesis in the absence of any exogenous treatment to damage DNA. Proc Natl Acad Sci USA 94:13792–13797.
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39. Yuzhakov A, Turner J, O’Donnell M (1996) Replisome assembly reveals the basis for asymmetric function in leading and lagging strand replication. Cell 86:877– 886. 40. Stukenberg PT, Studwell-Vaughan PS, O’Donnell M (1991) Mechanism of the sliding -clamp of DNA polymerase III holoenzyme. J Biol Chem 266:11328 – 11334. 41. Courcelle CT, Courcelle J (2006) Monitoring DNA replication following UV-induced damage in Escherichia coli. Methods Enzymol 409:425– 441. 42. Khlebnikov A, Datsenko KA, Skaug T, Wanner BL, Keasling JD (2001) Homogeneous expression of the P(BAD) promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter. Microbiology 147:3241–3247. 43. Delagoutte E, von Hippel PH (2001) Molecular mechanisms of the functional coupling of the helicase (gp 41) and polymerase (gp 43) of bacteriophage T4 within the DNA replication fork. Biochemistry 40:4459 – 4477. 44. Stan NM, et al. (2005) Nature 435:370 –373.
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