Helicase and polymerase move together close to the fork junction and copy DNA in one-nucleotide steps

SUMMARY

By simultaneously measuring DNA synthesis and dNTP hydrolysis, we show that T7 DNA polymerase and T7 gp4 helicase move in sync during leading strand synthesis taking one-nucleotide steps and hydrolyzing one dNTP per base-pair unwound-copied. The cooperative catalysis enables the helicase and polymerase to move at a uniformly fast rate without GC-dependency or idling with futile NTP hydrolysis. We show that the helicase and polymerase are located close to the replication fork junction. This architecture enables the polymerase to use its strand-displacement synthesis to increase the unwinding rate while the helicase aids this process by translocating along single-stranded DNA and trapping the unwound bases. Thus, in contrast to the helicase-only unwinding model, our results suggest a new model where the helicase and polymerase are moving in one-nucleotide steps and DNA synthesis drives fork unwinding and a role of the helicase is to trap the unwound bases and prevent DNA reannealing.

INTRODUCTION

Replicative helicases are ring-shaped hexameric proteins that unwind duplex DNA using the energy of NTP hydrolysis (Patel and Picha, 2000). In addition to DNA unwinding, replicative helicases play a key role in coordinating leading and lagging strand synthesis by mediating interactions with the polymerase and primase enzymes (Benkovic et al., 2001; Hamdan and Richardson, 2009; Indiani et al., 2009; Patel et al., 2011b). In isolation, replicative helicases are efficient single-stranded (ss)DNA translocases, but slow at unwinding the duplex DNA relying completely or partly on thermal fraying of the junction base pair (Donmez et al., 2007; Johnson et al., 2007; Kim et al., 2002; Lionnet et al., 2007; Ribeck et al., 2010) However, when the helicase is associated with the replicative polymerase, unwinding occurs at a fast rate (Dong et al., 1996; Kim et al., 1996; Korhonen et al., 2004; Manosas et al., 2012b; Stano et al., 2005). Similarly, replicative polymerases rely on helicases to processively copy the duplex DNA (Kang et al., 2012; Manosas et al., 2012b; Stano et al., 2005). The mutual dependency between the two enzymes is generally observed in replicative complexes from various organisms, but the structural and biochemical basis of the synergy is not fully understood.

In this paper, we address the central question of how helicase and polymerase synergistically increase the activities of DNA unwinding and synthesis during leading strand synthesis. Using T7 DNA helicase and T7 DNA polymerase as model replication enzymes, we address several questions essential to understanding the helicase-polymerase coupling mechanism: How is the stepping mechanism of the helicase coordinated with the single-nucleotide stepping mechanism of the DNA polymerase during leading strand synthesis? Where is the polymerase located with respect to the fork junction and the helicase in the leading strand complex? What is the role of strand-displacement synthesis activity of the polymerase? There is consensus that DNA polymerase elongates the primer one base at a time and therefore translocates with a step-size of one-nucleotide, but the chemical step-size (base pairs unwound per NTP hydrolyzed) of replicative helicases is not known. Therefore, we do not know how the helicase and polymerase coordinate their stepping mechanisms to stay together during leading strand synthesis. It is known that the physical coupling between T7 gp4 and T7 DNA polymerase is essential for processive DNA synthesis (Kulczyk et al., 2012; Zhang et al., 2011). However, there are no structures of leading strand complexes; therefore, we lack basic information such as where the helicase and polymerase are located in relation to the fork junction and whether the polymerase is directly or indirectly involved in increasing the unwinding rate. Most models assume that the helicase unwinds the DNA and the polymerase trails many nucleotides behind the helicase (Kulczyk et al., 2012; Liu et al., 2013) preventing helicase’s backward slips by DNA synthesis (Delagoutte and von Hippel, 2001; Stano et al., 2005). However, recent studies of T4 replication enzymes suggested that the polymerase might be actively involved in unwinding and helicase prevents fork regression and backward translocations of the polymerase (Manosas et al., 2012a; Manosas et al., 2012b).

Crystal structures of hexameric E. coli Rho helicase (with ssRNA and ATP analog) and papilloma virus replicative helicase E1 (with ssDNA and ADP) revealed that each subunit of the ring engages one-nucleotide of nucleic acid and binds to one NTP, which suggests one-nucleotide per NTP step-size (Enemark and Joshua-Tor, 2006; Thomsen and Berger, 2009), but a recent crystal structure of replicative DnaB helicase of E. coli shows two-nucleotides of ssDNA bound per subunit suggesting two-nucleotide per NTP step-size (Itsathitphaisarn et al., 2012). Ensemble unwinding studies have measured the kinetic step-size of T7 gp4 helicase as 10 bp and that of DnaB helicase as 1.4 bp (Galletto et al., 2004; Jeong et al., 2004). The variation in kinetic step-size may arise from various reasons, including heterogeneity in the reaction rates, helicase slippage, and different oligomeric forms (Lohman et al., 2008; Patel and Donmez, 2006). The kinetic step size estimates the number of base pair unwound between successive rate limiting steps and is not equivalent to the chemical step-size, which estimates the number of base pairs unwound per NTP hydrolyzed. To understand the coupling between polymerase and helicase stepping, we set out to determine the chemical step-size, which has not been convincingly determined for replicative helicases.

Most studies determine the chemical step-size from the ratio of base pair unwinding and NTP hydrolysis rates, but the challenge is making sure that the two rates are measured under the exact same conditions. Typically, to prevent DNA reannealing during unwinding, DNA concentrations are kept low (~ 1–5 nM). However, under low DNA concentrations, NTP hydrolysis rate is not accurately measured because of the small signal. Thus, when the two activities are measured separately using different DNA and helicase concentrations, there is a possibility that the active enzyme concentration is not the same in the two assays and the ratio is not an accurate measure of the chemical step-size. This explains the variation in the reported chemical step size from one to four base pairs per NTP (Donmez and Patel, 2008; Kim et al., 2002).

We have overcome these limitations by developing a one-pot assay that quantifies base-pair separation and NTP hydrolysis in a single reaction mixture, which has not been achieved before. We applied this method to determine the chemical step-size of the replicative helicase in complex with the polymerase using T7 gp4 and T7 DNA polymerase enzymes. The results show that for every base pair unwound and synthesized by the helicase-polymerase there is one dNTP hydrolyzed. Therefore, the chemical step-sizes of DNA synthesis and unwinding are perfectly coordinated during leading strand replication allowing the helicase and polymerase to move in sync. We show that together the helicase and polymerase unwind-copy the duplex DNA at a uniformly fast rate without GC-dependency or idling with futile NTP hydrolysis. To understand the structural basis of cooperativity between the helicase and polymerase, we mapped their locations at the replication fork with respect to the fork junction. In contrast to existing models, we find that helicase and polymerase are juxtaposed close to the fork junction, and in this configuration, the polymerase can unwind the junction base-pair during leading strand synthesis by strand-displacement synthesis. Our results support the new model of leading strand synthesis, where the helicase and polymerase enzymes are tracking close to the junction base pair and DNA synthesis is driving fork unwinding and the helicase is trapping the unwound bases and preventing DNA reannealing.

RESULTS

One-nucleotide chemical step-size of the helicase and polymerase

To determine the chemical step-size of unwinding during leading strand synthesis, we designed a new one-pot assay that determined how many base pairs of DNA are unwound per dNTP hydrolyzed by the helicase. Since processive strand displacement DNA synthesis by T7 DNA polymerase is dependent on the activity of T7 gp4 helicase (Stano et al., 2005), the rate of dNMP incorporation provides an accurate measure of the base-pair unwinding rate of the helicase (Pandey et al., 2010; Pandey et al., 2009). A major advantage of using DNA synthesis to measure unwinding is that the unwound strands cannot reanneal with DNA synthesis occurring simultaneously. Therefore, high DNA concentrations can be used, which allows accurate measurement of the dNTP hydrolysis kinetics. An important criterion for accurate determination of the chemical step-size is to make sure that dNTP hydrolysis and dNMP incorporation are measured simultaneously. This avoids uncertainties due to imprecise knowledge of active enzyme complex concentration. The one-pot assay we developed uses [α³²P] dNTPs to monitor both DNA synthesis and dNTP hydrolysis activities in a single reaction mix (Figure 1A). DNA synthesis is quantified from incorporation of radiolabeled dNMPs in the primer and dNTPase kinetics from radiolabeled dNDP production. If the reaction produces equimolar amounts of dNDPs as the dNMPs incorporated into DNA, it will indicate an average chemical step-size of one-nucleotide, whereas a 1:2 ratio of dNDP:dNMP will indicate a step-size of two-nucleotides.

Figure 1. Chemical step-size of unwinding during leading strand DNA synthesis.

(A) One-pot step size assay to measure the base pairs unwound and synthesized per dNTP hydrolyzed. T7 gp4 and T7 DNA polymerase bound to 60-bp replication fork (2 μM each) with dTTP and dATP (0.2 mM each) spiked with [α-³²P] dTTP and [α-³²P]dATP were reacted with MgCl₂ from milliseconds to seconds at 18^°C. (B) Representative thin layer chromatography (TLC) shows the separation of dTTP and dATP from corresponding dNDPs and DNA (at the origin) generated at increasing time on the 100% AT-rich replication fork. The TLC analysis enables simultaneous measurement of the kinetics of dNTP hydrolysis and DNA synthesis. (C) High resolution sequencing gel shows the kinetics of leading strand DNA synthesis on the 100% AT-rich fork DNA with the top panel showing total radioactivity in each lane. (D) Kinetics of total dNMPs incorporated into the 100% AT-rich fork DNA from the gel-based and TLC assays are plotted with the kinetics of total dNDPs produced by the helicase. (E) Kinetics of total dNMPs incorporated into the 50% GC-rich fork DNA and corresponding total dNDPs produced. (F) Fraction of individual dNTP hydrolyzed in 100% AT-rich experiment between 0.9 and 4 s of reaction. (G) Fraction of individual dNTP hydrolyzed in 50% GC-rich fork experiment between 0.9 and 5 s of reaction. Errors are ± standard error of mean (SEM). See also Figure S1 and Table S1.

T7 gp4 and T7 DNA polymerase were preassembled on the 60-bp long replication fork (100% AT-rich, Figure 1A, Table S1) in the presence of dTTP and dATP without Mg²⁺. The reactions were initiated with Mg²⁺ in a rapid quenching instrument and stopped after milliseconds to second time intervals before the entire 60-bp dsDNA region is unwound and copied. The 100% AT-rich fork requires only dATP and dTTP; therefore, all the substrates and products could be resolved on a single polyethyleneimine thin layer chromatography (TLC) plate. The highly charged DNA stays close to the origin, while the dNDPs and dNTPs migrate higher and are resolved (Figure 1B). The reactions were spiked with [α³²P] dTTP and [α³²P] dATP to quantify the molar amounts of dNTP hydrolyzed and DNA synthesized. The DNA synthesis rate was also determined independently by resolving the products on a sequencing gel (Figure 1C). After subtracting the background hydrolysis in the absence of Mg²⁺, (Supplemental Information) the molar ratio of dNDPs produced to dNMP incorporated into DNA is obtained (Figure 1D). The results from two independent kinetic experiments show that T7 gp4 and T7 DNA polymerase produce the same amount of dNDPs from dNTP hydrolysis as dNMPs incorporated into the DNA (Figure 1D and Figure S1B). This indicates that for every dNTP hydrolysis by the helicase, there is one base-pair unwound-synthesized. Since the polymerase incorporates dNMPs one base at a time, the coupling ratio of one indicates that the average chemical step-size of the helicase is also one-nucleotide. Thus, our results show for the first time that the polymerase and helicase are moving together taking one-nucleotide steps during leading strand synthesis.

One-nucleotide chemical step-size is maintained with the 50% GC-rich DNA

To determine how the increased GC-content in the DNA affects the chemical step-size and the synchronous movement, we measured DNA synthesis and dNTP hydrolysis on the 50% GC-rich replication fork (Figure 1E). These experiments require all four dNTPs that also needed to be resolved from the corresponding dNDPs. Therefore, we carried out the reaction in two parts: In one experiment, the reactions were spiked with [α³²P]-labeled dATP and dTTP, and in another, the reactions were spiked with [α³²P]-labeled dGTP and dCTP. The kinetics of total dNTP hydrolysis was determined form the two TLC analyses and compared to the kinetics of total dNMPs incorporated in the same time (Figure 1E and Figure S1C). The time courses of dNTP hydrolysis and dNMP incorporation overlay quite well indicating that the chemical step-size is one-nucleotide, irrespective of the GC-content. Thus, our results show that T7 gp4 does not slip or idle with dNTP hydrolysis when it is coupled with T7 DNA polymerase.

Fuel preference of the helicase

It is known that dTTP is the preferred substrate of T7 gp4 that supports efficient translocation and unwinding (Matson and Richardson, 1983; Sun et al., 2011). However, it is not known whether T7 gp4 maintains the same substrate specificity when it is working with T7 DNA polymerase. Our one-pot assay monitors the hydrolysis kinetics of individual dNTPs; therefore, it is an ideal set up to determine the nucleotide preference of T7 gp4 during leading strand synthesis (also see Supplemental Information). Analysis of each dNTP hydrolysis on both 100% AT-rich and 50% GC-rich fork experiments shows that dTTP is the preferred fuel of T7 gp4 (Figure 1F, 1G and Figure S1D, S1E). T7 gp4 also uses dATP but the dTTP:dATP utilization ratio is ~2:1. The nucleotide dGTP is used less frequently and dCTP is not used at all. These results indicate that the dTTP usage specificity of T7 gp4 is the same whether it is translocating on its own or with T7 DNA polymerase in the leading strand complex.

Uniform leading-strand DNA synthesis rate across the spectrum of GC-content

We have shown earlier that the unwinding rate of the isolated T7 gp4 helicase decreases from ~56 bp/s to ~7.6 bp/s with increasing GC-content (Donmez and Patel, 2008). Whether the unwinding-synthesis rates of helicase-polymerase are affected by increasing GC-content was not fully investigated (Pandey et al., 2009). To determine the GC-dependency, we measured the DNA synthesis rates on a series of replication forks (5, 20, 35, 50, 65, 80 %GC-content). The experiments were carried out by preincubating T7 gp4 and T7 DNA polymerase on the primer-labeled 40 bp replication fork with dTTP minus Mg²⁺ (Figure 2A) to synchronize the reactions, and then measuring the transient state kinetics of primer elongation after addition of Mg²⁺ (also see Supplemental Information). The formation and decay of each DNA of increasing length with time was globally fit into the polymerization model (Pandey et al., 2010; Pandey et al., 2009) to obtain the DNA synthesis rates (Figure S2).

Figure 2. Polymerase stimulates the leading strand synthesis rate across the spectrum of GC-content in the replication fork.

(A) T7 DNA polymerase and T7 gp4 (125 nM each) were preassembled on primer-labeled fork DNA (100 nM) with 0.5 mM dTTP and the reactions were initiated with MgCl₂ and 0.1 mM rest of the dNTPs and quenched after millisecond time intervals. The DNA products were resolved on sequencing gels (Figure S2) and single-nucleotide incorporation rates and errors were determined by fitting the data to the polymerization model. (B) The average rate of DNA synthesis by the helicase-polymerase on forks with different GC-content is compared with the base pair unwinding rates of the helicase on forks of the same sequence (Donmez and Patel, 2008). (C) The ratio of the DNA synthesis rate and helicase unwinding rate shows that the polymerase stimulates the unwinding rate to different extents depending on the GC-content. Errors are ± standard error of mean (SEM). See also Figure S2 and Table S1.

The analysis shows that the AT-rich replication forks are unwound and copied at an average rate of 90–100 nt/s whereas the GC-rich forks are unwound and copied at 70–80 nt/s (Figure 2B). This is only a 25% drop in rate, in contrast to nearly 10-fold drop in unwinding rate with helicase alone. Interestingly, T7 DNA polymerase accelerates the unwinding rate to different extents across the GC-content; the acceleration is about 2-fold on low GC and about 9-fold on high GC forks (Figure 2C). As a result of this differential rate enhancement, the final leading strand synthesis rate becomes almost independent of the GC-content. Taken together the results indicate that the helicase and polymerase enzymes are moving together at a nearly uniform rate through DNA of different GC-content.

Architecture of the helicase-polymerase at the replication fork

T7 DNA polymerase and T7 gp4 are bound to opposite strands of the replication fork but are physically coupled through specific interactions between the C-terminal tail of T7 gp4 and a front basic patch on the polymerase (Figure S6) that are important for maintaining processivity (Kulczyk et al., 2012; Liu et al., 2013). How the two enzymes are positioned at the replication fork with respect to the junction base pair is not known. There are three ways to physically couple the helicase and polymerase at the replication fork (Figure 3A): a) polymerase is behind the helicase (‘polymerase-trailing’ model), b) helicase is behind the polymerase (‘helicase-trailing’ model), and c) helicase and polymerase are both close to the fork junction (‘proximity’ model). In the polymerase-trailing model, the helicase is juxtaposed close to the fork junction and the polymerase is trailing behind the helicase separated from the fork junction by a gap of ssDNA. According to this model, only the helicase is unwinding the DNA and the trailing DNA polymerase can increase the unwinding rate by preventing backward slippages of the helicase. In the helicase-trailing model, the polymerase is close to the fork junction and the helicase is trailing behind separated by some bases from the fork junction. According to this model, the polymerase is unwinding the DNA and the helicase is preventing back slippages of the polymerase. In the proximity model, both helicase and polymerase are close to the fork junction. In this configuration, the polymerase and helicase can jointly unwind the junction base pair, trap unwound bases, and prevent each other’s backward slippages. Distinguishing between these models will provide structural insights for understanding the synergy between the helicase and polymerase. We therefore designed several experiments to determine the relative positions of the helicase and polymerase with respect to fork junction within the leading stand complex.

Figure 3. Exonuclease mapping for helicase location on the replication fork with the polymerase.

(A) Cartoons show three general models of helicase-polymerase coupling within the leading strand complex. The polymerase trailing model shows the helicase close to the fork junction and the DNA polymerase trailing behind the helicase separated by a gap of ssDNA (in red) from the fork junction. Helicase trailing model shows the polymerase close to the fork junction and the helicase trailing behind separated by a gap of ssDNA (in red). The proximity model shows that the DNA polymerase and helicase are both juxtaposed close to the fork junction. (B) Sequencing gel (bottom) shows the 3′ to 5′ exonuclease digestion of the 5%GC40dsFP fork (top) with its top strand labeled at the 5′-end with ³²Pi. The fork junction is designated as ‘0’, and downstream bases are labeled ‘+1’ onward. The middle panel shows peak analysis of the Hel-Pol lane. The ‘mm’ distance is from the uncut DNA (75-mer or +40 position). See also Figure S3 and Table S1.

Mapping the helicase position at the replication fork junction

We used exonuclease III + exonuclease T digestion to map the downstream boundary of T7 gp4 helicase in the leading strand complex with T7 DNA polymerase. Exo III digests duplex DNA stepwise in the 3′ to 5′ direction and exo T digests ssDNA in the same direction. Thus, combination of the two exonucleases of predetermined concentration (Figure S3A) enabled us to map the helicase position at the junction of duplex and ssDNA region on the replication fork. T7 gp4 and T7 DNA polymerase were assembled on the replication fork in the presence of a non-hydrolyzable dTTP analog, dTMPPCP, and subjected to exonuclease digestion. The helicase and polymerase position were mapped by labeling the lagging and leading strands, respectively (Figure S3B). If the downstream boundary of the helicase is at the fork junction, then the digestion products will be around 36-nt long (length of the ssDNA lagging strand), and if the helicase is trailing behind the polymerase, then the digestion products will be shorter. The digestion patterns of the lagging strand with helicase alone and helicase-polymerase are similar with a prominent 38-nt central band, which indicates that the downstream boundary of the helicase is at fork junction (position +1, Figure 3B). The mapping of the leading strand shows that the polymerase is bound to the primer-template junction under these conditions (Figure S3B). Replication forks without helicase and polymerase and with polymerase only are completely digested in the same time of exonuclease treatment. These results indicate that the leading edge of the helicase is close to the fork junction both in the absence and in the presence of the polymerase, which rules out the ‘helicase trailing’ model.

Location of the polymerase at the replication fork junction

We designed two experiments to determine the precise location of the T7 DNA polymerase relative to the fork junction in a strand-displacing complex with the helicase. In one experiment, we introduced a transplatin interstrand crosslink at the 17^th position from the fork junction in the duplex DNA (Figure 4A, Figure S4). Transplatin crosslinks GC base-pair without major DNA structural distortion; hence, it is a good choice to monitor the movements of the helicase-polymerase (Paquet et al., 1999). The interstrand DNA crosslink will stall the actively unwinding helicase, and at this point we can determine the precise location of the polymerase by measuring primer extension in millisecond time-scale and with single-nucleotide resolution. If the polymerase is trailing behind the helicase, then primer extension will stop/pause many nucleotides before the 17^th crosslinked base-pair (‘polymerase trailing’ Figure 3A). If the polymerase is traveling close to the fork junction, then primer extension will stop before the crosslinked base-pair (‘proximity model’ Figure 3A).

Figure 4. Mapping the polymerase location during leading strand synthesis using interstrand transplatin crosslinked fork DNA.

(A) DNA sequence of the fork with interstrand transplatin crosslink at the 17th GC base pair from the fork junction.(B) Sequencing gels show the kinetics of primer extension on unmodified (left) and transplatin crosslinked fork (right). Reactions were carried out at 18^°C using 150 nM T7 gp4,T7 DNA polymerase, 100 nM fork, 1 mM dNTPs each, and 4 mM MgCl₂. (C) Overlaying kinetics of DNA synthesis on unmodified and transplatin crosslinked fork DNA from the gels in the B panels. (D) Peak analysis of primer extension reactions on unmodified (left) and crosslinked fork (right) at 0.08 s, 0.3 s and 1 s from the gels in panel B. See also Figure S4 and Table S1.

Our results show that primer extension stops at the 16^th base, just before the crosslink (Figure 4B). Except for some pausing observed one and two nucleotides before the crosslink, there is no pausing at any of the earlier positions that would indicate that the polymerase is trailing behind the helicase or pushing the helicase out of the way (Figure 4B and 4D). The lack of slowing or pausing was confirmed by quantifying the primer extension rate on crosslinked and unmodified forks, which shows that leading strand synthesis rate proceeds at the same rate on both forks up to the 16^th nucleotide when the crosslink arrests DNA synthesis (Figure 4C). These results indicate that T7 DNA polymerase within the strand-displacing helicase-polymerase complex is tracking close to the fork junction. We observe this also with exo⁺ T7 DNA polymerase (Figure S4). These results indicate that the polymerase is tracking close to the fork junction and hence consistent with the ‘proximity’ model.

In the second type of mapping experiment, T7 gp4 was stalled at the fork junction with the non-hydrolyzable dTTP analog, dTMPPCP. T7 gp4 does not translocate in the presence of dTMPPCP and binds DNA with nM K_d and lifetime of hours (Hingorani and Patel, 1996; Kim et al., 2002; Sun et al., 2011). The polymerase was then walked towards the stalled helicase by primer extension that was initiated 16-nucleotides upstream of the fork junction (Figure 5A). The polymerase copies the ssDNA gap within milliseconds and stalls at the 16^th position, just one nucleotide before the fork junction (Figure 5B and 5C, left panels). Similar to the transplatin crosslink, there is a slight accumulation of product one to two nucleotides before the fork junction, but this is observed with and without the helicase (Figure 5C). Thus it appears that the polymerase is sensing the duplex DNA ~2 nucleotides before the fork junction. Quantifying the kinetics of each nucleotide addition up to the fork junction shows that polymerase copies the ssDNA gap all the way to the fork junction with an average rate of 264±48 nt/s without helicase and 213±53 nt/s with helicase, which indicates that the helicase presence at the fork junction does not hamper the polymerase from reaching the junction (Figure 5D). Interestingly, without helicase, the polymerase continues to catalyze strand-displacement synthesis ~6 nucleotides beyond the fork junction (Figure 5B, right panel). However, with the helicase, the polymerase goes only ~3 nucleotides beyond the fork junction (Figure 5B, left panel), which indicates that coupling between the helicase and polymerase stops DNA synthesis closer to the fork junction. Collectively, the two mapping experiments rule out the ‘polymerase-trailing’ model and indicate that the helicase and polymerase are close together at the junction as in the ‘proximity’ model.

Figure 5. Kinetics of polymerase walking towards a stalled helicase at the fork junction.

(A) The fork DNA was designed with 16-nt ssDNA gap between the primer end and the fork junction. T7 gp4 and T7 DNA polymerase (150 nM each) were preincubated on the replication fork (100 nM) with dTMP-PCP (100 μM), the non-hydrolyzable analog of dTTP, and reactions were initiated with 1 mM dNTPs at 18^°C. In the cartoon, we show no interactions between helicase-polymerase, but the two may interact by looping out the single-stranded template in the gap. In another experiment, helicase was left out. (B) Sequencing gels show the primer extension kinetics on helicase-stalled fork (left) and without helicase (right). The arrows show the position of the junction base pair on the fork. (C) Peak analysis of gels in panel B at 0.4 s of reaction on helicase-stalled fork (left) and without helicase (right) (D) The single-nucleotide incorporation rates on helicase-stalled fork (green bar) and without helicase (purple bar) are compared up to the fork junction. Errors are ± standard error of mean (SEM). See also Table S1.

Role of polymerase in DNA unwinding

The proximity of the polymerase to the fork junction raises the possibility that the polymerase can also unwind the DNA during leading strand synthesis through its strand-displacement synthesis activity, which would explain the increase in unwinding rate with the polymerase. It is known that T7 DNA polymerase can catalyze strand-displacement synthesis on its own with a low processivity (Stano et al., 2005) and the processivity is increased in the presence of E. coli single strand binding (SSB) protein (Nakai and Richardson, 1988). What is not known is if the strand-displacement activity of the polymerase itself is fast enough to support leading strand synthesis. To address this question, we measured the rate of strand-displacement synthesis rate using transient state kinetic experiments. T7 DNA polymerase was preassembled on a 50% GC-rich replication fork substrate and the progressive primer elongation reaction was measured in the millisecond time scale with and without E. coli SSB (Figure 6A). We chose E. coli SSB because of its high affinity for ssDNA (Syed et al., 2014) as compared to T7 single strand binding protein gp2.5 (Rezende et al., 2002). Also unlike gp2.5, there are no known interactions of E. coli SSB with T7 DNA polymerase. In the absence of SSB, T7 DNA polymerase strand displaces and copies only five base pairs of duplex DNA (5%GC40ds fork). Adding a protein trap did not change the kinetics of strand displacement synthesis indicating that the reason for limited strand displacement by the polymerase is not due to its dissociation from the DNA (Figure S5). The first two junction base pairs are copied with an average rate of 141±24 nt/s and the remaining are copied at a slower rate (Figure 6B, blue bars). With SSB, the polymerase copies the entire duplex with an average rate of 145±46 nt/s (Figure 6B, yellow bar), which is faster than the leading strand synthesis rate with the helicase (114±25 nt/s) (Pandey et al., 2009) (Figure 6B, green bar). These results demonstrate that the polymerase on its own can unwind the duplex DNA at a fast rate through strand displacement DNA synthesis (Figure 6B and Figure S5), but it needs SSB to trap the displaced strand consistent with the idea that fork reannealing limits the strand displacement synthesis activity of the polymerase (Manosas et al., 2012a). The results suggest that helicase may act in a similar way; that is, trap the unwound bases through its ability to bind tightly to ssDNA. Being a motor which is physically coupled to the polymerase, the helicase can track with the polymerase and locally trap the bases in a step-wise manner. In this manner the helicase can also modulate the strand-displacement activity of the polymerase.

Figure 6. Kinetics of strand-displacement synthesis by the polymerase.

(A) Representative sequencing gels show the kinetics of strand-displacement synthesis by T7 DNA polymerase alone (left panel) and polymerase with E. coli SSB (right panel) at 18ºC. The 50% GC-rich fork (100 nM) was preincubated with the polymerase (100 nM) and reactions were initiated with dNTPs (1 mM) with and without SSB (1 μM). (B) The single-nucleotide incorporation rates of T7 DNA polymerase (blue bar) across the duplex region are compared with rates of polymerase+SSB (yellow bar) and polymerase+helicase (green bar) (Pandey et al., 2009). Errors are ± standard error of mean (SEM). See also Figure S5 and Table S1.

DISCUSSION

The results of our study provide further insights into the mechanism of synergistic unwinding and DNA synthesis by the helicase and polymerase during leading strand synthesis. By tracking the polymerase’s movement through DNA synthesis and helicase’s movement through its dNTPase activity, we demonstrate that the two motors are moving in sync at the replication fork and pulling the two strands of the fork DNA at the same rate. The one-to-one correspondence between the kinetics of dNTP hydrolysis and dNMP incorporation shows for the first time that T7 gp4 helicase and polymerase are moving in steps of one-nucleotide. The single molecule studies of T7 helicase also provide evidence for stepping behavior of T7 helicase consistent with 1:1 coupling between stepping and hydrolysis (Syed et al., 2014). The single base pair chemical step size is consistent with the crystal structures of the hexameric helicases E1 and Rho, where each subunit spans one nucleotide of DNA or RNA and binds one ATP (Enemark and Joshua-Tor, 2006; Thomsen and Berger, 2009). The one-nucleotide step size is an emerging theme in helicases and one can imagine that one advantage of single base pair unwinding step-size would be speed, in that it would allow the enzymes to capitalize on thermal breathing fluctuations of the junction base pair for fast fork movement (Jose et al., 2012). Our results also indicate that when coupled with T7 DNA polymerase, the helicase does not slip or idle with dNTP hydrolysis. Interestingly, the single molecule studies are suggesting that the isolated T7 helicase has backward movements but only at low dTTP concentrations (Syed et al., 2014).

T7 DNA polymerase and T7 gp4 are physically coupled during leading strand synthesis and this coupling is important for maintaining high processivity (Kulczyk et al., 2012; Zhang et al., 2011). However, in the absence of synchronous stepping, one motor can outrun the other creating ssDNA loop between the helicase, polymerase, and fork junction. It is known that T7 gp4 unwinds the DNA past the stalled polymerase, albeit at slow rates (Patel et al., 2011a). This indicates that when the polymerase is stalled, there will be ssDNA loop between the polymerase and the fork junction, which has been proposed as a signal to recruit DNA repair proteins at the replication fork (Byun et al., 2005). Interesting, the polymerase does not go very far past the stalled helicase (Figure 5B, left panel). This indicates some scrunching but no extensive ssDNA loop or gap created between the stalled helicase and fork junction. This probably has biological significance such as preventing aberrant DNA synthesis.

Most models in the literature position the helicase close to the replication fork junction and the polymerase many nucleotides behind the helicase (‘polymerase-trailing’ model) (Kulczyk et al., 2012; Liu et al., 2013). Although the models do not clearly define the separation, the polymerase could be >10 nucleotides behind the fork junction, but there is no evidence for this configuration. Our exonuclease mapping experiments show that the helicase is positioned at the fork junction in the leading strand complex with the polymerase (Figure 3 and Figure S3). Similarly, using transplatin interstrand DNA crosslink, we show that the polymerase synthesizes DNA right up to the crosslink (Figure 4). Some pausing was observed 1–2 nt before the fork junction, which might indicate that the polymerase needs 1–2 nt of ssDNA ahead. Collectively our results indicate that the helicase and polymerase are proximal during normal replication (‘proximity’ model, Figure 3A). However, there could be flexibility in the physical coupling; that is, the two may interact by looping out the ssDNA gap or when the helicase or polymerase encounters a problem along the way, ssDNA loop is created to enable one to move past the other.

Having the polymerase and helicase tracking close to the junction base pair during normal replication provides an elegant mechanism for synergistic unwinding and DNA synthesis, now observed in many systems (Dong et al., 1996; Kim et al., 1996; Manosas et al., 2012b; Patel et al., 2011b; Stano et al., 2005). Previous models assumed that the helicase is primarily responsible for unwinding the fork junction and the polymerase increases the unwinding rate by trapping the unwound bases by DNA synthesis (Stano et al., 2005). The data presented in this paper indicate otherwise. We show that T7 DNA polymerase on its own can unwind the fork junction at a fast rate, but in the absence of the helicase, DNA synthesis is limited to a few base pairs. Single molecule studies of T4 polymerase showed that the main cause for limited strand-displacement synthesis activity of the polymerase is fork regression (Manosas et al., 2012a). This is consistent with our results that show that E. coli SSB with its ability to simply bind the ssDNA without unwinding (Meyer and Laine, 1990) supports fast and processive DNA synthesis by T7 DNA polymerase (Figure 6). Stimulation of strand-displacement activity of polymerase by SSB has been observed in other systems (Cha and Alberts, 1989; Soengas et al., 1995; Yuan and McHenry, 2009). Interestingly, the strand-displacement rate of T7 DNA polymerase with SSB is 1.3 times faster than the leading strand synthesis rate with T7 gp4 helicase. One might ask why replicative complexes need a helicase motor to trap ssDNA, when SSB can serve this function. Replicative helicases mediate key interactions with the leading and lagging strand polymerases and the primase enzyme, which is critical for coordinating the synthesis of the two strands. In addition, as a motor that is physically coupled to the polymerase, the helicase can locally trap the bases in a step-wise manner and modulate the strand-displacement activity of the polymerase to control the rate of leading strand synthesis (Pandey et al., 2009).

What is the mechanism of DNA unwinding by the replicative DNA polymerase? It is clear that DNA synthesis is required to stimulate unwinding, which means that somehow nucleotide addition and translocation are coupled to base pair unwinding. Crystal structures of DNA polymerases consistently show a sharp kink in the DNA template between the templating n base and the next n+1 base (Figure S6) (Doublie et al., 1998). As a consequence of template DNA kinking, the n+1 base is unpaired and flipped out. This must occur after each cycle of dNMP addition and PPi release, when the polymerase transitions from the pre-translocated state to the post-translocated state. During this process, as shown in Figure 7, the n base occupies the templating position in the active site of the polymerase and the n+1 base is flipped from the neighboring. Thus, pre- to post-translocation transition and DNA kinking can provide a specific mechanism for unwinding the junction base pair. There is biochemical and structural evidence that RNA polymerases utilize such a mechanism to unwind the DNA one base pair ahead of the 3’-end of the primer (Kashkina et al., 2007; Yin and Steitz, 2002, 2004). Unlike RNA polymerases, however, most replicative polymerases lack a mechanism to prevent reannealing of the unwound bases, a role fulfilled by the associated helicase that traps the complementary bases by translocating on the lagging strand. We propose that replisomes of bacteria, mitochondria, and phages where the helicase and polymerase are bound to opposite strands of the replication fork use this general mechanism to couple DNA unwinding to DNA synthesis (Dong et al., 1996; Korhonen et al., 2004; Manosas et al., 2012b; Patel et al., 2011b; Stano et al., 2005). However, eukaryotic replicative helicases such as MCM (Mini Chromosome Maintenance) that unwind DNA in the 3′-5′ direction are expected to bind to the same strand as the polymerase and work in a different manner.

Figure 7. Model of leading-strand DNA synthesis.

The helicase and polymerase are juxtaposed close to the fork junction in the leading strand complex interacting with each other via the basic patch at the front edge of the polymerase and the acidic C-terminal end of T7 gp4 (Kulczyk et al., 2012; Zhang et al., 2011). We propose that in a single cycle of dNMP addition by the polymerase, the helicase hydrolyzes one dNTP. The junction base-pair is unpaired as dNTP binds and the polymerase pulls in the template base n (in blue) and flips the next base n+1 (in red). The helicase captures the unpaired base (in red) as its leading subunit (E-state) transitions into the dNTP bound T*-state. At the same time, a series of reactions around the gp4 ring result in dNTP tightening (T*→T), dNTP hydrolysis (T→DP), Pi release (DP→D), dNDP release (D→E) at the rest of the subunits to release a nucleotide base at the trailing end of the helicase (D-state, light blue subunit). ‘*’ represents weak binding (Patel et al., 2011b). See also Figure S6.

EXPERIMENTAL PROCEDURES

One pot assay to measure the chemical step-size during leading strand synthesis

T7 gp4 (gp4A’ with M64L mutation to prevent translation of gp4B) and T7 gp5 (exo-) proteins were purified as described (Patel et al., 1992; Patel et al., 1991). The 60-bp replication fork was assembled by annealing three complementary DNAs (Table S1 and Supplemental Experimental Procedures). T7 gp4 and T7 DNA polymerase (2 μM each final) were preassembled on the 100% AT-rich 60-bp replication fork with dTTP and dATP (0.2 mM each, final) spiked with [α-³²P] dTTP and [α-³²P]dATP in the replication buffer (50 mM Tris Cl pH 7.6, 40 mM NaCl, 1 mM DTT and 10% glycerol). The reactions were initiated with MgCl₂ at 18°C using RQF-3 Rapid Quench-Flow Instrument (KinTek Corporation) and stopped after ms to s with formic acid (4M). Similar experiments with the 50% GC fork were performed with 0.2 mM each of all four dNTPs in two parts, one with radiolabeled dTTP and dATP and another with radiolabeled dGTP and dCTP. Radiolabeled dNTPs and dNDPs were resolved from each other and the nascent radiolabeled DNA on PEI-Cellulose F thin layer chromatography (TLC) plate developed in 0.5 M–0.6 M potassium phosphate buffer pH 3.4. The neutralized reactions were also applied to a high resolution polyacrylamide/7 M urea/1.5 x TBE sequencing gel. The TLCs and gels were exposed to the phosphorimager screens, scanned, and the products were quantitated using the ImageQuant software. A control experiment was carried out without MgCl₂ to correct for background dNTP hydrolysis. Total dNDPs and dNMPs added in DNA were determined from the following equations:

$$\text{total}[\text{dNDPs}]\mathrm{\mu }\mathrm{M}=(\text{counts}{(\text{dNDPs})}^{\ast }400\mathrm{\mu }\mathrm{M})/\text{counts}(\text{dNDPs}+\text{dNTPs}+\mathrm{D})$$ equation 1

$$\text{total}[\text{dNMPs}\text{in}\text{DNA}]\mathrm{\mu }\mathrm{M}=(\text{counts}{(\mathrm{D})}^{\ast }400\mathrm{\mu }\mathrm{M})/(\text{dNDPs}+\text{dNTPs}+\mathrm{D})$$ equation 2

Where counts(dNDPs), counts(dNTPs) and counts(D) are added dNDPs, added dNTPs, and DNA, respectively. In experiments with 50% GC fork, total [dNDPs] from the two TLC were added together. Total [dNDPs] were subtracted from background total [dNDPs] from a control experiment without Mg²⁺, and the corrected total [dNDPs] and total [dNMPs] were plotted as a function of reaction time (Figure 1D and 1E). The kinetics of individual dNTP hydrolysis was determined as described in the Supplemental Experimental Procedures.

Transient state kinetics of DNA synthesis

The transient state kinetics of DNA synthesis was measured using the rapid quenched-flow instrument at 18°C. The platinated intercrosslinked duplex DNA fork was prepared following published methods (Dalbies et al., 1995; Dalbies et al., 1994) as outlined in Supplemental Experimental Procedures. All other DNA forks were prepared by annealing complementary oligonucleotides listed in Table S1 (also see Supplemental Experimental Procedures). T7 gp4 and T7 DNA polymerase or polymerase alone was preassembled on the replication fork (with either [γ³²P] or fluorescein 5′ labeled Primer 24) in the presence of dTTP in replication buffer and mixed with dATP, dCTP and dGTP, and MgCl₂ to initiate reactions. The EDTA-quenched (300 mM) reactions were resolved on 24% acrylamide/7M urea/1.5xTBE sequencing gel. The kinetics were globally fit to the polymerization model using MATLAB and mgfit (see gfit.sourceforge.net) to obtain the single-nucleotide incorporation rate constants.

Exonuclease mapping

A typical reaction contained Exo III and Exo T (1 U/μl each), 5%GC40dsFP DNA fork DNA (50 nM), T7 gp4 (50 nM), T7 DNA polymerase (50 nM), dTMP-PCP (100 μM), DTT (1 mM) and MgCl₂ (10 mM) in replication buffer incubated for 2–20 min at 30 °C. The EDTA-quenched (300 mM) reactions were resolved on 24% acrylamide/7M urea/1.5xTBE sequencing gel.

HIGHLIGHTS.

• DNA unwinding occurs in steps of one-nucleotide during leading strand synthesis.

• Helicase-polymerase unwind-copy different GC-content DNA at a uniformly fast rate.

• Leading strand polymerase and helicase are juxtaposed, close to the fork junction.

• The polymerase itself can catalyze fast strand-displacement synthesis at the fork.