RecA acts as a switch to regulate polymerase occupancy in a moving replication fork
Contributed by Mike O'Donnell, February 19, 2013 (sent for review February 6, 2013)
Abstract
This report discovers a role of Escherichia coli RecA, the cellular recombinase, in directing the action of several DNA polymerases at the replication fork. Bulk chromosome replication is performed by DNA polymerase (Pol) III. However, E. coli contains translesion synthesis (TLS) Pols II, IV, and V that also function with the helicase, primase, and sliding clamp in the replisome. Surprisingly, we find that RecA specifically activates replisomes that contain TLS Pols. In sharp contrast, RecA severely inhibits the Pol III replisome. Given the opposite effects of RecA on Pol III and TLS replisomes, we propose that RecA acts as a switch to regulate the occupancy of polymerases within a moving replisome.
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Cells contain several different translesion synthesis (TLS) DNA polymerases (Pols) that can synthesize DNA across damaged nucleotides, but TLS Pols are typically low fidelity and mutagenic (ref. 1 and references therein). In Escherichia coli, TLS Pol II and TLS Pol IV are present in normal growing cells along with the high-fidelity chromosomal replicase, Pol III. Previous studies have shown that all three Pols function at replication forks with DnaB helicase, primase, the β sliding clamp, and the clamp loader (2). Bulk chromosome replication is performed by Pol III, and the mechanism that regulates the access of TLS Pols to the replisome is largely unexplored.
Heavy DNA damage in E. coli induces the SOS response, which slows down chromosomal replication (3) and expresses over 40 proteins that aid cell survival and enable genome replication to continue (4, 5). Induction of the SOS response requires formation of ssDNA by replication forks to which the cellular recombinase, RecA, binds. The binding of RecA to ssDNA facilitates RecA-mediated self-cleavage of the LexA repressor, thereby activating the SOS response genes (6). Among these are TLS Pol II (polB) and TLS Pol IV (dinB), which are rapidly (<5 min) up-regulated during the SOS response (7–10). TLS Pol V (UmuD′2C) is induced late (≥45 min) and requires RecA bound to ssDNA (RecA*) for synthetic activity (8, 11, 12). Pol IV and Pol V belong to the Y-family Pols (1), whereas Pol II, even though involved in TLS and mutagenesis, is a B-family polymerase (13).
During normal chromosome duplication, the intracellular levels of Pols II and IV are not sufficient to take over Pol III-based replisomes (2). Upon DNA damage, the induced levels of TLS Pols take over the cellular replicase (2, 14, 15). Our previous studies have shown that Pols II and IV replace Pol III within the replisome without causing fork collapse, as they preserve the DNA-bound helicase and sliding clamp to form TLS Pol replisomes (2). Pol II and Pol IV replisomes progress at significantly slower rates than the intrinsic rate of DnaB helicase, and thus regulate the speed of helicase unwinding. Slower fork progression may give the cell additional time to repair DNA lesions using normal repair processes (e.g., nucleotide excision repair), thereby preventing direct encounters of the replication fork with DNA lesions.
It has been suggested that polymerase selection at the replication fork is mainly guided by mass action dictated by the concentration of each polymerase and its affinity for the clamp (1, 14, 16, 17). However, the findings of this report demonstrate that RecA adds an additional level of control, acting as a switch that regulates DNA polymerase access to the replication fork. Surprisingly, RecA strongly inhibits fork progression by Pol III replisomes, yet RecA stimulates TLS Pol II and Pol IV replisomes. The mechanism that underlies the opposite effects of RecA on Pol III and the TLS Pol replisomes involves single-strand binding protein (SSB). Although all of the polymerases function with SSB on the template strand, SSB inhibits TLS Pol function in the context of a replisome, presumably acting in trans by binding lagging-strand ssDNA. RecA relieves the SSB induced repression of TLS Pol replisomes and stimulates their action, while inhibiting the Pol III replisome. We propose that the opposite effects of RecA on Pol III and TLS Pol replisomes facilitate the switch of TLS Pols with Pol III during the cellular response to DNA damage.
Results
RecA Inhibits the Pol III*-Replisome.
The cellular replicase, Pol III*, is composed of three Pol III cores attached to the three τ subunits of the clamp loader [(Pol III)3τ3δδ′χψ] (18–20) (Fig. 1A). One Pol III core functions on the leading strand, whereas the other two Pol III cores extend multiple Okazaki fragments on the lagging strand (20, 21). The τ subunits also connect to the hexameric DnaB helicase that encircles the lagging strand, forming a replisome that we refer to as the Pol III*-replisome in this report (i.e., Pol III*-β-DnaB). Primase acts distributively and periodically targets the helicase for activity, ensuring that RNA primers are located near the replication fork (22).
Fig. 1.
To examine the effect of RecA on the Pol III*-replisome, we used a 100-mer synthetic rolling circle DNA (Fig. 1A). The leading-strand template (inner circle) contains only dG, dC, dA, enabling the leading strand (and not the lagging strand) to be labeled using [α-32P]dTTP. The DnaB helicase is first assembled onto the 5′ ssDNA tail in a 2-min preincubation, followed by assembly of the Pol III*-β clamp in the absence of dTTP to prevent forward progression during a further 4-min incubation. DNA synthesis is initiated upon adding [α-32P]dTTP, the four ribonucleotide triphosphates (rNTPs), primase, and SSB. When present, RecA is added 2 min before initiating DNA synthesis, unless indicated otherwise. Timed aliquots are withdrawn and DNA products are analyzed in an alkaline agarose gel.
The results of these assays show that RecA greatly inhibits leading-strand synthesis by the Pol III*-replisome (Fig. 1B). To determine whether RecA inhibition occurs during elongation or initiation, we tested the effect of adding RecA after DNA synthesis has been initiated (Fig. 1C). The result shows that RecA also inhibits a moving Pol III*-replisome. Thus, RecA up-regulation during the SOS response should inhibit fork progression by Pol III*-β. Our earlier studies on the effect of RecA on Pol III*-β have also demonstrated that RecA can disassemble the replicase from DNA (23). Furthermore, a recent in vivo study using single-molecule techniques finds that RecA indeed binds to DNA at the replication fork, and favors disassembly of the Pol III replisome (24).
We wanted to understand how RecA inhibits the replication fork. We note that SSB is highly stimulatory to the Pol III*-replisome (Fig. 1B). Hence, one possibility is that RecA competes with SSB, indirectly inhibiting the Pol III*-replisome. Recently, it has been shown in vivo that RecA induces Pol III dissociation from the fork in a RecF, RecO, and RecR (RecFOR)-independent manner (24). Although RecFOR promote efficient exchange of RecA for SSB at blocked forks (refs. 25–27 and references therein), shorter RecA-filaments are formed in absence of RecFOR and are proposed to bind lagging-strand ssDNA produced during fork advance (24). Extension of RecA filaments toward the replisome destabilizes the Pol III*-replisome, facilitating its dissociation, consistent with our earlier in vitro observations that RecA dislodges Pol III* from a primed site (23). Another possibility is that RecA inhibits a component of Pol III*, either Pol III core, the clamp loader, or the τ subunit of the clamp loader.
In Fig. 2, we examined the effect of RecA on Pol III core-replisomes lacking the τ subunit. Elimination of the τ subunit from the reaction is made possible by the fact that the dnaX gene encoding τ also encodes a smaller subunit, γ (28). The τ subunit can be replaced by γ within the clamp loader without effect on clamp-loading activity (29). However, unlike τ, the γ subunit cannot bind Pol III core and DnaB helicase. Without the connection between Pol III core and DnaB helicase, the Pol III core-replisome is much slower than the Pol III*-replisome (30). In addition, because the clamp loader contains three copies of either τ or γ (31), when γ replaces τ, leading- and lagging-strand polymerases are no longer connected together by the clamp loader. The effect of RecA on the Pol III core-replisome, assembled using Pol III core, β and the γ complex (Fig. 2A), shows that RecA no longer has a significant effect on the Pol III core-replisome (Fig. 2B). The absence of an effect by RecA indicates that RecA does not interfere with either Pol III core or the clamp loader.
Fig. 2.
To test whether RecA interferes with the τ function, we added extra τ into Pol III*-replisome reactions containing RecA. Addition of excess τ only slightly inhibits the Pol III*-replisome (lanes 1 and 2). In contrast, excess τ partially restores activity of the Pol III*-replisome in the presence of RecA (Fig. 2C, lanes 3 and 4). We have not detected a physical interaction between τ and RecA, although such an interaction could be weak and hard to detect. The τ subunit is also known to bind ssDNA, and therefore RecA may compete with τ to bind ssDNA, decreasing the affinity of Pol III* to DNA and ejecting Pol III* from the fork. Pol III*-replisomes and Pol III core-replisomes also differ by the fact that leading- and lagging-strand synthesis are performed simultaneously by the Pol III*-replisome, but are physically uncoupled in replisomes containing Pol III core (i.e., τ is needed to connect Pol III cores together). This unique multipolymerase feature of Pol III* raises the possibility that RecA inhibits the lagging-strand polymerase within the Pol III*-replisome and prevents forward motion of the leading polymerase. We tested an effect of RecA on lagging strand synthesis in two ways. In one, we tested the effect of RecA on Pol III*-β using single primed M13 ssDNA, a lagging-strand model template (Fig. S1B). The result shows that RecA only inhibits Pol III*-β by about 50%, suggesting that it does not shut down Pol III action on the lagging strand. As a more definitive test, we omitted primase from a Pol III*-replisome reaction and observed that RecA still inhibits the leading-strand polymerase, even with no lagging-strand synthesis. RecA inhibition of the replisome in the absence of primase indicates that RecA inhibition is not based in coupled leading/lagging replication (Fig. S2).
The observation that RecA does not inhibit the Pol III core-replisome, but strongly inhibits the Pol III*-replisome, led us to examine the effect of SSB. The result shows that SSB inhibits the Pol III core-replisome (Fig. 2D), contrary to the observed SSB stimulation of the Pol III*-replisome (Fig. 1B). SSB inhibition of Pol III core-β conflicts with the known stimulatory effect of SSB on Pol III core-β activity on a primed ssDNA template (32). Stimulation of Pol III core-β by SSB is also shown in Fig. S1C under the conditions used in this report. The fact that Pol III core-β is normally stimulated by SSB that is on the same strand as the polymerase, yet SSB inhibits the leading-strand Pol III core-β in the context of a replisome, suggests that SSB inhibits the leading-strand Pol III-core-β by acting in trans through binding lagging-strand ssDNA (discussed further, below).
RecA Activates TLS Pol Replisomes.
Next, we examined the effect of RecA on the Pol II-replisome (Fig. 3A). Surprisingly, RecA greatly stimulates Pol II leading-strand synthesis (Fig. 3B, lanes 1–3 and 7–9). In contrast, SSB strongly suppresses Pol II-replisome activity (lanes 1–3 and 4–6). SSB even inhibits the moving Pol II-replisome when it is added after initiation (Fig. S3). Furthermore, Pol II-replisomes in the presence of RecA show comparable leading-strand replication with or without SSB (Fig. 3B, lanes 4–6 and 10–12). Taken together, these data suggest that Pol II-replisomes function in the presence of RecA, that SSB inhibits replication, and that RecA overcomes SSB inhibition of the Pol II-replisome.
Fig. 3.
Pol II-β is active on primed ssDNA where SSB is bound to the template strand (i.e., stimulated by SSB, not inhibited) (7, 33), as confirmed here using primed M13 ssDNA coated with SSB (Fig. S4). Therefore, SSB inhibition of the leading-strand Pol II-replisome in Fig. 3B is not the expected result if SSB were binding the leading strand. Indeed, SSB is not thought to enter the leading strand of a replisome (34). However, the lagging strand is known to create ssDNA for SSB binding. Hence, we presume that SSB inhibition is due to SSB binding the lagging-strand ssDNA and acting in trans to repress the leading-strand Pol II [SSB directly interacts with Pol II (35)]. We also find that RecA interferes with Pol II activity on M13 ssDNA with and without SSB, unlike RecA stimulation on the leading strand (Fig. S4B). The fact that both RecA and SSB bind ssDNA, and that their effect on Pol II at a fork is opposite to their effect on Pol II on primed ssDNA is consistent with the hypothesis that SSB and RecA exert their action on the leading polymerase in trans by binding the lagging-strand ssDNA. An alternative explanation is that SSB inhibits DnaB helicase activity, although this possibility seems unlikely given the large stimulatory effect of SSB on the Pol III*-replisome. To test this possibility, we examined DnaB activity in the presence and absence of SSB, but observed no inhibition of helicase action by SSB (Fig. S5).
In Fig. 3C, we examine the effects of RecA and SSB on the Pol IV-replisome. The Pol IV-replisome is less active in the presence of SSB (Left gel) and RecA largely overcomes the SSB inhibition (Right gel lanes 1–6). Hence the Pol IV-replisome behaves similarly toward SSB and RecA as the Pol II-replisome. Pol IV-β synthesis on primed M13 ssDNA demonstrates that Pol IV-β functions well with and without SSB (Fig. S4C, lanes 1–4), and RecA does not substantially affect its activity in the presence of SSB (Fig. S4C, lanes 5 and 6). However, when SSB is omitted, Pol IV is strongly inhibited by the presence of RecA (Fig. S4C, lanes 7 and 8).
We also examine TLS Pol V function in the context of a replisome (i.e., at a replication fork). It is well established that Pol V requires RecA bound to ssDNA (RecA*) (12). In Fig. 3C, lanes 7–12, we test the Pol V-replisome in the presence and absence of RecA*. The result shows that Pol V is capable of functioning within a replisome with β and DnaB, but, as expected, it requires RecA. Consistent with the other TLS polymerases, the Pol V-RecA*-replisome is inhibited by SSB (Fig. S6).
TLS Pols Rapidly Exchange Within the Replisome.
We previously showed that TLS Pols II and IV exchange with Pol III* during replisome progression, although RecA was not present in those studies (2). According to our recent findings, one may presume that, once RecA has been induced, Pol III* is no longer capable of function. Thus, it becomes more relevant to understand whether different TLS Pols trade places with one another within a TLS Pol-replisome in the presence of RecA. In Fig. 4A, we examine whether Pols II and IV can trade places within a moving replisome in the presence of RecA. The results show that as Pol IV is titrated into a Pol II-replisome reaction, the replisome slows down. Because the Pol IV-replisome is much slower than the Pol II-replisome, this observation indicates that Pol IV is capable of exchange with Pol II within a moving TLS Pol replisome.
Fig. 4.
It is important to note that all of the replication forks migrate as a single population, suggesting that TLS Pols II and IV exchange with one another quite rapidly at the fork (rapid within the timescale of the fork speed). Specifically, if a mixture of the two different TLS Pol-replisomes were processive, distinct populations representing each replisome speed would be observed. The presence of a single population of replisome moving at a rate that is intermediate between the speeds of either polymerase by themselves, reveals that the TLS Pols II and IV freely exchange with one another during fork progression. We also examined whether Pol V switches with Pol II and Pol IV during TLS Pol-replisome progression. Switching of TLS polymerases during fork movement is indeed the case, as illustrated by the slower rate of synthesis of the Pol II and Pol IV-replisomes when Pol V is titrated into the reactions (Fig. 4 B and C).
Rapid exchange of TLS Pols within moving TLS Pol-replisomes would ensure that when a replication fork encounters a lesion, each TLS Pol in the cell will have access to the lesion, thereby enabling the most capable TLS Pol for the particular lesion to enter the resplisome and traverse the lesion.
Primase Functions in the Context of TLS Pol-Replisomes in the Presence of RecA.
The results thus far have focused on the leading strand. Hence it remains possible that RecA inhibits priming activity and prevents lagging-strand synthesis. The synthetic rolling circle template enables specific labeling of either strand, depending on whether [α32P]dATP or [α32P]dTTP is added. In Fig. 5, we examine leading- and lagging-strand synthesis by the Pol II- and IV-replisomes (Fig. 5 A and B). Considering that both TLS Pols are present in the cell, we also tested a mixture of Pols II and IV (Fig. 5C). As expected, both strands are made in approximately equal amounts. Alkaline agarose gel analysis reveals that the Okazaki fragments are 100–200 bp for the TLS Pol-replisomes, much shorter than the 1–2 kb for the Pol III*-replisome (36) (Fig. 5D). This result is not surprising given the stochastic action of primase (22, 37), which may be expected to synthesize primers at shorter intervals for slow moving versus fast replisomes. Overall, the results of Figs. 4 and 5 indicate that TLS Pols rapidly exchange in the replisome, that primase is active in the presence of RecA, and that both leading and lagging strands are replicated by TLS Pol-replisomes.
Fig. 5.
Discussion
This report demonstrates that RecA has a global impact on E. coli DNA polymerases at replication forks. Both in vivo and in vitro studies indicate that TLS Pols can switch with the Pol III replicase during replication and that the switching process mainly occurs by simple mass action (2, 15, 17). Although mass action may be an important determinant, most likely the process is more complex (14, 38). In eukaryotes, TLS polymerase switching correlates with DNA damage-induced ubiquitination of the proliferating cell nuclear antigen clamp and phosphorylation of key replisome proteins (39–42). Although bacterial processes are expected to be simpler than in a eukaryote, our data add an additional layer of control on the switching process beyond simple mass action. Specifically, the results presented here demonstrate that RecA acts as a master switch that regulates polymerase exchange at the fork (Fig. 6). Thus, RecA inhibits the chromosomal replicase, Pol III*, and activates the TLS Pol II, Pol IV, and Pol V for function with DnaB, primase, and the β sliding clamp.
Fig. 6.
A surprising finding is that RecA strongly inhibits leading-strand advance by the Pol III*-replisome, and this finding is supported by a recent in vivo study that visualizes RecA binding and dissociation of Pol III (24). The precise mechanism underlying RecA inhibition could occur in any of several possible ways. RecA inhibition of the Pol III*-replisome requires τ (Fig. 2), which connects Pol III* to the helicase and is essential for cell viability (30, 43). RecA may interact directly with τ and inhibit it, perhaps by competing with τ for ssDNA and expelling Pol III* from the replisome. Another possibility is that RecA may compete with SSB for lagging-strand ssDNA. Although RecA binding to SSB-coated ssDNA is presumed to require RecFOR in vivo, there are at least two conceivable ways in which RecA may bind ssDNA without RecFOR. One possibility is that the replisome may contain a RecA-loading activity, similar to that found in RecBCD, which loads RecA as it proceeds to unwind DNA (44). Another possibility is that the moving replisome may enable RecA to bind to the unwound ssDNA before SSB, due to the small binding site size of RecA. Specifically, only 3–5 nt are needed for RecA, whereas 35–65 nt are required for each SSB tetramer (45–48). This second possibility has also been suggested by a recent study that directly visualizes fluorescent RecA at the fork in live cells even in the absence of RecFOR (24). As the fork advances, generating lagging ssDNA, RecA may get the first chance to bind ssDNA until the ssDNA achieves a long enough length to bind SSB. At this point, SSB presumably binds and dislodges RecA. However, a nucleation cluster of RecA could be formed, and with sufficient intracellular amounts of RecA, it may expand (49). Even if only a transient small patch of RecA exists, it would be located right next to the leading-strand polymerase and may exert an influence on it. Moreover, the RecA nucleation center would be constantly replenished while earlier RecA clusters are dismantled by SSB as the fork progresses. This proposed dynamic would have a second-order kinetics that depends on the relative concentrations of SSB, RecA, and their intrinsic rates of association with ssDNA. In the presence of τ, forks move at a rapid pace and may quickly produce the proper site size of ssDNA for SSB binding. Without τ, Pol III core forks move much slower and could give more time for RecA to bind ssDNA before ssDNA reaches a sufficient length for SSB to bind. A deeper understanding of these parameters at a moving replication fork clearly awaits further studies.
In sharp contrast to RecA inhibition of the Pol III*-replisome, RecA stimulates TLS Pol replisomes. The mechanism of this opposite effect could be based in the different response of these replisomes to RecA and SSB. Specifically, SSB inhibits leading-strand synthesis by the Pol II- and Pol IV-replisomes, and RecA overcomes this inhibition. Neither Pol II nor Pol IV is inhibited by SSB on primed ssDNA templates, and in fact Pol II is stimulated by SSB. Hence, it would appear that SSB does not inhibit TLS Pol-replisomes by binding the leading strand, but instead by binding the lagging strand and altering the leading polymerase activity. One may speculate that SSB on the lagging strand directly contacts the polymerase on the leading strand and inhibits its action. In fact, direct contact of SSB to Pol II and to Pol IV has been documented (35, 50). RecA may overcome SSB inhibition by simply occupying the lagging strand adjacent to the leading polymerase and prevent SSB binding (Fig. 6).
Several genetic data support these biochemical findings. Studies of the recF,O,R genes imply RecA regulates TLS Pol function, and the phenotypes may be explained by RecA-mediated regulation of TLS Pol access to replication forks (51). Moreover, it has been shown that replication forks slow down in response to DNA damage, when RecA and TLS Pols are produced (3). In vivo overexpression of Pol IV greatly slows DNA replication (2, 15). Additional genetic studies also indicate that Pols II and IV gain access to the replication fork in a RecA-mediated fashion (52). The authors demonstrate that, in the dnaN159 lexA51(Def) strain, the growth defect is suppressed by inactivation of the dinB-encoded Pol IV. Introduction of the recA730 allele into the same strain yields a synthetic lethal phenotype that is suppressed by inactivation of the polB-encoded Pol II or the umuDC-encoded Pol V (52). These observations are consistent with RecA facilitating takeover of rapid Pol III*-replisomes by slow TLS Pols. Furthermore, Pol IV is known to perform −1 frameshifts that are suppressed by UmuD (uncleaved form) in a RecA-dependent manner (53). Hence, yet other proteins may be expected to modulate the actions of Pol IV, in addition to RecA.
Materials and Methods
Materials.
Subunits of the Pol III replicase (α, ε, θ, γ, τ, δ, δ′, χ, ψ, and β), Pol III core (α, ε, and θ), Pol IV, DnaB, primase, SSB, γ complex (γ3δδ′χψ), and τ complex (τ3δδ′χψ) were reconstituted and purified as previously reported (2, 54). Pol III* [(Pol III core)3τ3δδ′χψ] was reconstituted and purified as described (54). Pol V, RecA, and 30-mer ssDNA were obtained as previously described (12). Pol II containing a His6 tag was cloned into pET16, expressed, and purified by Ni2+ affinity chromatography; its activity was the same as that of wt Pol II. The 100-mer rolling circle DNA was assembled as described (2). ϕX174 ssDNA (New England Biolabs) is annealed to a 5′ 32P-labeled DNA 70-mer (40 5′ dT nt tail and 30 nt annealed to ϕX174). Buffer A is 20 mM Tris·Cl (pH 7.5), 0.1 mM EDTA, 5 mM DTT, 5% (vol/vol) glycerol, 40 μg/mL BSA, and 8 mM MgCl2. Reaction buffer (Buffer R) is 20 mM Tris (pH 7.5), 8 mM MgCl2, 5 mM DTT, 0.1 mM EDTA, 25 mM sodium glutamate, and 4% (vol/vol) glycerol.
Replication Reactions.
DnaB was assembled on the synthetic rolling circle DNA upon preincubation of 100 fmol DNA with 4 pmol DnaB6 and 50 μM ATPγS in 15 μL of Buffer A for 2 min at 37 °C. Then, Pol III* (4 nM), β (14 nM), and 60 μM each of dGTP, dCTP, dATP were added and incubated a further 4 min. Because leading-strand synthesis requires dTTP, fork movement is prevented. DNA synthesis was initiated upon adding 1 μg of SSB, 50 μM each of the four rNTPs, 60 μM DnaG primase, 0.5 mM ATP, and 20 μM dTTP in a 25-μL final reaction volume. When present, RecA is added 2 min before initiating DNA synthesis, unless indicated otherwise. DNA synthesis was monitored using [α-32P]dTTP (leading strand) or [α-32P]dATP (lagging strand) (3,000–5,000 cpm/pmol). Pols II, IV, and/or V were added as indicated in the figure legends. Reactions lacking Pol III* contained 140 nM Pol III core or 130 nM Pol II or 200 nM Pol IV or 500 nM Pol V, 2 nM τ (or γ)-complex, and 14 nM β. Reactions using activated RecA (1.0 μM RecA) included a preincubation of 14.4 μM RecA with 1.4 μM 30-mer ssDNA and 0.71 μM ATPγS for 5 min at 37 °C in 52.5 μL of Buffer R. Reactions were quenched upon addition of 25 μL of 1% SDS/40 mM EDTA. One-half of the reaction was analyzed on 0.6% alkaline agarose gels, and DNA synthesis was quantitated by processing the other half on DE81 filters and liquid scintillation as described (54).
Helicase Assays.
DnaB was preloaded on ϕX174 ssDNA to which was annealed a DNA 70-mer with a 5′ 32P-dT40 ssDNA tail in a 5-min preincubation with 50 μM ATPγS at 37 °C. Substrate unwinding was initiated by adding ATP, and SSB (when present). Enzyme reactions (12 μL) contained 2 nM DNA, 500 nM DnaB, 50 μM ATPγS, 5 mM ATP, 1 μg of SSB, 5 mM creatine phosphate, 20 μg/mL creatine kinase, 20 mM Tris⋅acetate, pH 7.5, 10 mM MgCl2, 20% glycerol, 40 μg/mL BSA, and 5 mM DTT (pH 7.5). Reactions were quenched, and DNA products were analyzed on a native 10% PAGE (90 mM Tris⋅HCl⋅borate, 2 mM EDTA) using a Typhoon phosphorimager.
Acknowledgments
The authors are grateful for National Institutes of Health Grants GM39939 (to M.O.) and GM21422 and ESO12259 (to M.G.) in support of this project.
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Published online: March 18, 2013
Published in issue: April 2, 2013
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Acknowledgments
The authors are grateful for National Institutes of Health Grants GM39939 (to M.O.) and GM21422 and ESO12259 (to M.G.) in support of this project.
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The authors declare no conflict of interest.
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