Abstract
Most replicative helicases are hexameric, ring-shaped motor proteins that translocate on and unwind DNA. Despite extensive biochemical and structural investigations, how their translocation activity is utilized chemo-mechanically in DNA unwinding is poorly understood. We examined DNA unwinding by G40P, a DnaB-family helicase, using a single-molecule fluorescence assay with a single base pair resolution. The high-resolution assay revealed that G40P by itself is a very weak helicase that stalls at barriers as small as a single GC base pair and unwinds DNA with the step size of a single base pair. Single ATPγS binding could stall unwinding, demonstrating highly coordinated ATP hydrolysis between the six identical subunits. We observed frequent slippage of the helicase, which is fully suppressed by the primase DnaG. We anticipate that these findings allow a better understanding on the fine balance of thermal fluctuation activation and energy derived from hydrolysis.
Introduction
Helicases are essential enzymes for all life forms and catalyze the separation of double-stranded nucleic acids (dsNA) into single-stranded nucleic acids (ssNA), and many of these enzymes involved in DNA repair, DNA recombination and transcription termination are linked to human diseases (Crampton et al., 2006; Enemark and Joshua-Tor, 2008, 2006; Gai et al., 2004; Johnson et al., 2007; Lionnet et al., 2007; Lohman et al., 2008; Manosas et al., 2009; Pandey et al., 2009; Patel and Picha, 2000; Rasnik et al., 2006b; Ribeck et al., 2010; Rothenberg et al., 2007; Singleton et al., 2000; Thomsen and Berger, 2009; Wang et al., 2008; Yodh et al., 2010). Helicases are categorized into different superfamilies (SF) by protein sequence motifs, polarity of translocation and function among other criteria (Berger, 2008). Non-hexameric helicases with a pair of RecA-like domains are members of SF I and SF II, and are involved in DNA maintenance including repair, Holliday junction migration, chromatin remodeling, RNA melting and RNA-binding protein displacement. Hexameric or dodecameric helicases are classified into SF III-VI and are key players in DNA replication and transcription termination. SF III and SF VI helicases share a common fold called the AAA+ fold and members of the SF IV and V share the RecA-like fold. Both folds are structurally members of the ASCE (additional strand conserved E) superfamily of enzymes that consists of various multimeric enzymes with extremely diverse function (Erzberger and Berger, 2006).
Forward translocation of ring-shaped helicases on nucleic acids lattice is promoted by ATP hydrolysis. Based on crystal structures, two distinct models have been proposed concerning how the hydrolysis of ATP molecules in the multi-subunit enzyme is coordinated between the subunits: a sequential, ‘staircase-like’ model for ATP hydrolysis and a concerted ATP hydrolysis model. The staircase model based on the structures of BPV E1 helicase bound to ssDNA and E. coli Rho helicase bound to ssRNA (Enemark and Joshua-Tor, 2006; Thomsen and Berger, 2009) entails sequential hydrolysis of ATP around the hexameric ring, and one nt translocation for every ATP hydrolyzed. By extension, the unwinding step size has been proposed to be 1 base pair (bp) but this has not been experimentally tested. The concerted hydrolysis model based on the all-or-nothing nucleotide occupancy of SV40 Large T antigen structures (Gai et al., 2004) posits that the six ATP binding sites fire simultaneously, moving on the DNA by an increment determined by the stroke size of the DNA binding motif, which can be larger than 1 nt or 1 bp. For T7 gp4 helicase-primase, structural and ensemble kinetic data (Crampton et al., 2006; Liao et al., 2005; Singleton et al., 2000) suggested a sequential hydrolysis mechanism during DNA translocation, but the estimated unwinding step size is either larger than 1 bp (Johnson et al., 2007) or is variable depending on the GC content of the duplex DNA (Donmez and Patel, 2008; Syed et al., 2014). For the Rho helicase proposed to move in 1 nt steps (Thomsen and Berger, 2009), chemical interference data suggest that Rho needs to reset itself after it unwinds about ∼7 bp (Schwartz et al., 2009). For DnaB, ensemble kinetic studies supported a sequential ATP hydrolysis mechanism (Roychowdhury et al., 2009) with an unwinding step size of 1 bp (Galletto et al., 2004), but DnaB structure bound to ssDNA showed that one subunit of DnaB hexamer binds two nucleotides, leading to the proposal that DnaB unwinds DNA in two base pair steps (Itsathitphaisarn et al., 2012). Conflicting data and models call for experiments with sufficient spatio-temporal resolution to detect the elementary steps of unwinding. In the most comprehensive analysis of stepping by a ring-shaped motor on DNA, the DNA packaging motor from f29 was shown to package dsDNA in a hierarchy of non-integer, 2.5 bp steps, pausing after packaging 10 bp (Moffitt et al., 2009).
Results and Discussion
G40P unwinds dsDNA in single base pair steps
We probed the helicase activity of the phage SPP1 G40P, a DnaB type hexameric helicase (Berger, 2008; Pedré et al., 1994; Wang et al., 2008) required for phage replication in its bacterial host, using an unwinding assay (Ha et al., 2002; Myong et al., 2007; Pandey et al., 2009; Syed et al., 2014; Yodh et al., 2009) based on single-molecule FRET (Ha et al., 1996). The substrate is a 40 bp duplex DNA with 3’ and 5’ single stranded poly-dT tails, both 31 nt long, to mimic a replication fork, and is immobilized to a polymer-passivated surface via a biotin-neutravidin linker (Fig. 1a). FRET between the donor (Cy3) and the acceptor (Cy5) fluorophores conjugated to the fork was used to follow individual DNA unwinding in real time (Figure 1 – Figure Supplement 1). Duplex unwinding increases the time-averaged distance between the fluorophores therefore causing a reduction in FRET, and unwinding completion results in the release of the donor-labeled strand from the surface and an abrupt disappearance of total fluorescence (Fig. 1b and Figure 1 – Figure Supplement 1). Initial experiments were carried out using a DNA substrate with all AT base pairs (40 bp) and typical unwinding trajectories displayed a smooth and rapid FRET decrease at 1 mM ATP (Fig. 1c). Interestingly, when we repeated the experiment with a single GC bp inserted in the 11th position in otherwise all AT sequences (Fig. 1a), a large fraction (65 %) of unwinding trajectories showed a stall before full unwinding, with a characteristic stall lifetime of 79 ± 5 ms (Fig. 1d and Figure 1 – Figure Supplement 2a). FRET efficiencies of the stalled state were sharply distributed around EFRET= 0.52 ± 0.04 (blue lines in Fig. 1g), indicating that the stall occurs after a well-defined number of base pairs, presumably ten, have been unwound and is caused by a single GC base pair.
Individual traces revealed helicase slippage events, where partial unwinding is reverted before another unwinding attempt is made (Fig. 1d, lower panel). The lag time between successive unwinding attempts is less than 1 s on average, which is about 20-fold shorter than the de novo unwinding initiation time (see Methods and Figure 1 – Figure Supplement 1d), implying that the same enzyme is responsible for multiple partial unwinding and slippage events (Ha et al., 2002; Sun et al., 2011). In most slippage events followed by another unwinding attempt, the FRET efficiency returned to its original high value of DNA itself, indicating that the helicase slips backwards at least 10 base pairs.
DNA with 2 GC bp (11th and 12th bp) showed stalls at two distinct FRET levels (EFRET = 0.53 ± 0.04 and EFRET = 0.39 ± 0.04) determined from Gaussian fitting of EFRET distribution of stalled states (Fig. 1g). Because the first stall occurred at the same FRET level as observed with 1 GC bp, we attribute the second stall to the second GC bp. Two GC bp reduced full unwinding events to 57% of the traces.
Complete unwinding trajectories were even more rare (12%) for the DNA with 3 GC bp (11th, 12th and 13th bp). Stalls were identified from all traces, predominantly from unsuccessful unwinding attempts and are distributed in three distinct FRET levels with two main peaks centered at EFRET = 0.52 ± 0.04 and EFRET = 0.38 ± 0.04 and a small peak at EFRET = 0.27 ± 0.04 (Fig. 1g). The aforementioned crystallographic structures on ring-shaped helicases suggest step sizes between one and six nucleotides, resulting from a staircase like or spring-loaded or concerted ATP hydrolysis model (Enemark and Joshua-Tor, 2006; Gai et al., 2004; Itsathitphaisarn et al., 2012). Recent experimental studies have reported for hexameric helicases step sizes from one to three nucleotides depending on the GC content of the template (Syed et al., 2014). For our experimental design, we introduced GC barriers at the positions 11, 12 and 13. For a single GC barrier and a ring-shaped helicase with a step size of one nucleotide, we would expect the barrier-induced stalling at a single FRET level, corresponding to a helicase at position –1 relative to the GC base pair. If the helicase would unwind dsDNA with a step size of two nucleotides, and an unknown starting position on the DNA grid, we would expect to observe 50% of the helicases to stall at position –2 and 50% at position –1. In this case, we would observe two different FRET levels for a single GC base pair (Fig. 2a). Introducing a second GC base pair would lead in the case of a helicase with one nucleotide step size to two possible stalling events, one at position –1 and one at position 1 (Fig. 2b). Again, for a helicase with a step size of two nucleotides, we would expect two GC base pairs induce stalls at positions –2, –1 and 1 (Fig. 2b). Similarly, we could expect for three consecutive GC base pairs breaks at three positions for a step size of a single nucleotide and breaks at four positions for a step size of two nucleotides (Fig. 2c).
One, two or three discrete FRET values of stalled states in our data induced by one, two or three GC base pairs suggest that unwinding occurs in single base pair steps. As a further test, we obtained a calibration curve between the number of base pairs unwound and the FRET efficiency by performing the reaction using four different DNA constructs that have m AT base pairs followed by (40-m) GC base pairs (m = 4, 7, 10 and 13), which stalls the unwinding reaction presumably at the boundary between AT and GC base pairs (Fig. 1h). The stall FRET levels from the one, two and three consecutive GC base pairs fall within error in single base pair steps on our calibration curve (see Methods and Figure 1 – Figure Supplement 2). Therefore, we conclude that G40P unwinds DNA with a single base pair step size. Recent optical trap studies of the nonhexameric helicase HCV NS3 and XPD observed the long anticipated single base pair steps during unwinding (Cheng et al., 2011; Qi et al., 2013). Here, we extend direct observation of single base pair steps during DNA unwinding to the hexameric helicases.
The GC base pair induced pauses of the helicase imply that unwinding depends at least partially on the thermal fraying of the base pairs due to the higher thermodynamic stability of GC vs AT base pairs. Raising the experiment temperature increased the percentage of full unwinding events for the three GC bp construct, i.e. 62 % vs. 12 % at 33 ºC vs. 21 ºC, respectively (Table S2 and Figure 1 – Figure Supplement 2b).
Host primase DnaG prevents slippage events
A replicative helicase that cannot easily overcome GC base pairs would be ineffective. Because DnaG, the host primase, stimulates ATPase and unwinding activities of DnaB and G40P (Bird et al., 2000; Wang et al., 2008), we tested the effect of DnaG by forming a complex of G40P with the Bacillus subtilis primase DnaG in a ratio of 1:3 (Wang et al., 2008) before adding the complex to the immobilized DNA to initiate unwinding. Fig. 3a shows typical unwinding traces of the substrate with 3 GC bp in presence of the primase DnaG. Strikingly, the yield of complete unwinding increased significantly from 12% without DnaG to 58% with DnaG (Fig. 3b and Table S1). DNA molecules showing full unwinding did not show any slippage events, suggesting a novel function of DnaG that stimulates unwinding by preventing slippage. Presence of other replication proteins may further assist the helicase unwinding activity (Stano et al., 2005).
Slippage was also observed using the 40 AT DNA but less frequently (Fig. 3b). Because T7 gp4, E1 and Rho helicase have higher affinities to ssNA in the nucleotide-bound state (Adelman et al., 2006; Enemark and Joshua-Tor, 2006; Hingorani and Patel, 1993; Thomsen and Berger, 2009) we hypothesized that slippage would occur more frequently at lower ATP concentrations. Indeed, at sub-saturating ATP concentrations we observed a significant increase of slippage events (Fig. 3c) and the number of slippage events before complete unwinding increased for decreasing ATP concentrations (Fig. 3d). Inclusion of ADP in the reaction did not reduce or increase the slippage events significantly, indicating that the enzyme binds tightly to the DNA in both ATP and ADP bound states. However, presence of DnaG significantly reduced the average number of slippage events per full unwinding (≈2.5 fold) compared to G40P alone at all tested ATP concentrations (Fig. 3d, black triangles and Table S3).
ATP hydrolysis between the subunits of G40P is highly coordinated
G40P slippage can thus be reduced by keeping some nucleotides bound during each translocation step. To elucidate the ATP hydrolysis coordination between subunits, we included ATPγS in the reaction while keeping constant the combined concentration of ATP and ATPγS at 1 mM. ATPγS is a slowly hydrolysable analogue of ATP, and both nucleotides have nearly identical dissociation constants for G40P binding as tested using Mant-ADP (see Methods and Figure 3 – Figure Supplement 1). As expected, the unwinding rate of 40 bp, kunw, decreased as the ATPγS concentration increased (Figure 3 – Figure Supplement 1). Close examination of the unwinding traces with 2.5% ATPγS showed a pronounced stalling event that was rarely detected without ATPγS (Fig. 4a). The stall during unwinding was observed at broadly distributed FRET levels (Fig. 4e), suggesting the stall occurs stochastically, not at a particular location on DNA. At elevated ATPγS (≥ 20% ATPγS) no unwinding reaction was observed. The lifetime of the first stall during unwinding was 0.44 s ± 0.01 s, independent of ATPγS percentage (Fig. 4c), suggesting that the stall occurs when the enzyme is bound by a well-defined number of ATPγS. If a single ATPγS molecule is responsible for the hexamer stalling, the fraction of unwinding traces showing a stall event should depend linearly on the ATPγS concentration whereas a quadratic dependence is expected if two ATPγS molecules are necessary to stall the enzyme and so on. The stalled fraction increased linearly between 0.125% and 1% ATPγS (Fig. 4d, Figure 3 – Figure Supplement 1 and Methods), indicating that the stall is caused by a single ATPγS. A random ATP hydrolysis mechanism as observed for ClpX, a AAA+ protein unfolding machine (Martin et al., 2005), can thus be excluded for G40P. The model of a concerted ATP hydrolysis is very unlikely to apply to G40P considering the following results of our experiments: (i) The unwinding rate kunw versus ATP relation followed the Michaelis-Menten relation with a Hill coefficient close to 1, indicating that per enzymatic cycle only one ATP has to bind the enzyme (see Methods). (ii) Our slippage data indicate that an ATP or ADP free helicase loses grip to the tracking strand and slips backward. A concerted ATP hydrolysis leads to an ATP/ADP free helicase and an elevated probability to slip backwards at every unwound bp, which would be highly inefficient. Sequential nucleotide hydrolysis during ssNA translocation by hexameric helicases is also well supported by structural analysis (Enemark and Joshua-Tor, 2006; Thomsen and Berger, 2009) and was also observed in translocation experiments with T7gp4 (Crampton et al., 2006). A recent optical tweezers study on T7gp4 proposed a sequential nucleotide hydrolysis after analyzing the slippage probability at various nucleotide conditions (Sun et al., 2011). Here we show the first direct evidence for sequential hydrolysis during DNA unwinding.
How translocation leads to unwinding
Combining our data with the strand exclusion model of unwinding (Ahnert and Patel, 1997) and the staircase model of ssDNA translocation (Enemark and Joshua-Tor, 2006) we propose the following unwinding model for G40P that is likely to apply to other hexameric helicases. In the ATP and ADP bound state, G40P has a high affinity to the tracking strand. After ADP is released from one subunit the affinity of a central DNA-binding loop to the DNA backbone is reduced (Fig. 5a) (Enemark and Joshua-Tor, 2006; Thomsen and Berger, 2009). This loop then moves and contacts the backbone phosphate of the next base pair to be unwound when an ATP binds to the subunit. If the next base pair is an AT base pair, it melts rapidly and the relief of the structural distortion within the enzyme catapults the protein forward by one base pair. Subsequent ATP hydrolysis and ADP release repeats this scenario and the helicase steps in 1 bp/nt increments forward. However, in the case of a GC base pair, the melting step may be significantly slower, preventing a second DNA-binding loop from contacting the next base pair to be unwound because of steric constraints within the narrow central channel (Fig. 5b). Here, two alternative outcomes may be possible: (i) the helicase can move forward after slow melting of the GC base pair (step 3 top) or (ii) the other subunits of the helicase undergo ATP hydrolysis and product release, thereby losing contact with the tracking strand such that the helicase slips backwards and the DNA rezips until a new set of contacts is established to the tracking strand (step 3 bottom). For other helicases with wider central channels, the helicase may continue to step forward on the tracking strand without having to wait for duplex melting and may lead to unwinding of multiple base pairs in a burst after several nt translocation as has been proposed for a non-hexameric helicase (Myong et al., 2007).
At low ATP concentrations, the unbound DNA-binding loop moves forward to the next binding site. However, due to reduced ATP occupancy for this subunit, the loop would have difficulty in binding the next backbone position (Fig. 5c). Further ATP hydrolysis in the remaining subunits may lead to a situation of all DNA-binding loops being detached from the tracking strand. In this case, the helicase slips backwards and the DNA rezips again to its original conformation. DnaG has to interact tightly with single-stranded DNA in order to synthesize primers for the lagging strand. Such interactions would hinder slippage of G40P, promoting unwinding activity (Fig. 5d), maybe even by an induced conformational change in the helicase. In the case of ATPγS poisoning, G40P showed long stalling events. In such a case, a DNA-binding loop of the subunit bound by ATPγS cannot detach from the tracking strand due to the high affinity induced by the bound ATPγS (Fig. 5e). Thus, the helicase remains at the same position until ATPγS is released from the subunit.
Materials and Methods
Single-molecule FRET experiments and data analysis
Single-molecule FRET experiments were performed on a custom-built fluorescence microscopy setup and recorded with an EMCCD camera (Andor) with a time resolution of 30–100ms using custom C++ software. Single-molecule fluorescence traces were extracted by means of a custom IDL software.
Biotin was attached at the 5’ end of the DNA strand during DNA synthesis (Integrated DNA technologies). Cy3 N-hydroxysuccinimido (NHS) ester and Cy5 NHS ester (GE Healthcare) were internally labeled to the dT of single-stranded DNA strands by means of a C6 amino linker (modified by Integrated DNA Technologies, Inc.). A detailed list of all DNA strands used can be found in Table S4. A quartz microscope slide (Finkenbeiner) and coverslip were coated with polyethylene glycol (m-PEG-5000; Laysan Bio Inc.) and biotinylated PEG (biotin-PEG-5000; Laysan Bio Inc.) (Ha, 2001). Measurements were performed in a flow chamber that was assembled as follows. After the assembly of the coverslip and quartz slide, a syringe was attached to an outlet hole on the quartz slide through tubing. All the solution exchanges were performed by putting the solutions (100μL) in a pipette tip and affixing it in the inlet hole, followed by pulling the syringe. The solutions were added in the following order. Neutravidin (0.2 mg mL−1; Pierce) was applied to the surface and washed away with T50 buffer (10 mM Tris-HCl, pH 8, 50 mM NaCl). Biotinylated DNA (about 50–100 pM) in T50 buffer was added and washed away with imaging buffer (20 mM Tris-HCl, pH 8, 50 mM NaCl, 0.1 mg mL−1 glucose oxidase, 0.02 mg mL−1 catalase, 0.8% dextrose, in a saturated (∼ 3 mM) Trolox solution) (Rasnik et al., 2006a). For most experiments [ATP]= 1mM (except for the ATP titration experiments), G40P [hexamer]= 60nM in imaging buffer was injected to the flow chamber with 10mM MgCl2 while recording a fluorescence movie. For DnaG experiments we performed G40P/DnaG experiments by incubating G40P hexamers together with DnaG in a molar ratio 1:3, respectively. This mixture was then injected to the flow chamber in imaging buffer containing 60nM G40P hexamer concentration, 180nM DnaG monomer concentration, [ATP]=1mM and 10mM MgCl2. All experiments were performed at room temperature T=296 K.
FRET efficiency values (EFRET) were calculated as the ratio between the acceptor intensity and the total (acceptor plus donor) intensity after subtracting the background. Unwinding initiation times, unwinding times and stall lifetimes were scored by visual inspection of donor and acceptor intensities (Pandey, 2009). The stall FRET levels were averaged after visual scoring the stall lifetime of at least 5 data points. The number of slippage events before successful unwinding was measured by counting the number of crossings of a threshold EFRET = 0.7 –0.8 after box averaging the traces with a time resolution t=150ms. All data were analyzed and plotted with scripts written in MATLAB (Mathworks) and in Igor Pro (Wavemetrics).
DNA substrates
All DNA oligos were purchased from Integrated DNA Technologies (Coralville, IA) and site-specifically labeled with NHS-Cy3 or NHS-Cy5 obtained from GE Healthcare (PA13101 and PA15101, respectively; Pittsburg, PA). Table S4 lists all sequences. \iAmMC6T\ denotes the amine-modified thymine with a C6 spacer used for site-specific labeling. \5BiosG\ denotes the 5’ modification with biotin, \3Bio\ denotes the 3’ modification with biotin and \iCy5\ denotes the internal modification with Cy5 fluorophore.
Design of the unwinding substrate
The exclusion model of unwinding for hexameric helicases suggests that the tracking strand passes through the central pore of the hexameric protein and the non-tracking will be excluded. Thus, a labeling of the tracking strand might affect the unwinding reaction since the fluorophore would have to pass through the central channel. We tested this possibility by comparing average unwinding times of a substrate with labels at the tracking and non-tracking strand (Figure 1 – Figure Supplement 1a) as well as with labels just at the non-tracking strand (Figure 1 – Figure Supplement 1b). The average unwinding time of both substrates at [ATP]=1mM agreed very well within error (tavg(tracking+non-tracking) = 0.57±0.22s and tavg(double-labeled non-tracking) = 0.59±0.21s). However, the accessible FRET range in the case of the double-labeled non-tracking strand was significantly smaller (FRET efficiencies between 0.3 and 0.7, Figure 1 – Figure Supplement 1b) in contrast to the labeling at both DNA strands (FRET efficiencies between 0.95 and 0.11, Figure 1 – Figure Supplement 1a). A larger FRET efficiency range allows a higher resolution. Combined without detectable hindrance of the fluorophore passing through the central pore of G40P, further experiments were conducted with the substrate where both DNA strands labeled (Figure 1 – Figure Supplement 1a).
Unwinding initiation time depends linearly on the protein concentration
Figure 1b and Figure 1 – Figure Supplement 1c show typical unwinding traces after adding a solution containing G40P and 1 mM ATP at time tinject to the imaging chamber with immobilized DNA molecules. Unwinding begins after a delay (FRET starts to decrease as indicated by a decrease in donor signal and a concomitant increase in acceptor signal), which we call the unwinding initiation time, tinit. Unwinding itself takes a finite amount of time, tunw, during which FRET decreases to the lowest level and the total fluorescence signal disappears. The average tinit ranged from 6.6 s to 88 s as the protein concentration (in hexamer) is varied and is much longer than the average tunw ≈ 0.55 s at saturating [ATP] = 3mM. Therefore, unwinding events can be attributed to the action of a single functional unit of the helicase, which we presume to be a hexamer. The unwinding initiation rate, defined as the inverse of average tinit, increased linearly with protein concentration (Figure 1 – Figure Supplement 1d). In contrast, the average tunw did not show a significant dependence on protein concentration (inset Figure 1 – Figure Supplement 1d). The linear relation between the protein concentration and the initiation rate indicates that G40P is loaded as a preassembled hexamer instead of being assembled on the DNA in situ. Further support for the hexamer loading is provided by additional experiments blocking the free ssDNA end of the forked DNA with anti-digoxigenin (sketch in Figure 1 – Figure Supplement 1e). At [ATP] = 1mM and [G40P6] = 60nM, the percentage of single-molecule FRET traces that show unwinding events was reduced from 42% to 4.7% after incubation of the digoxigenin modified DNA construct with [anti-digoxigenin]=0.1mg/mL followed by washing out of unbound anti-digoxigenin (Figure 1 – Figure Supplement 1e).
ATP titration with ADP as competitive inhibitor
As expected, lowering the ATP concentration, from 3 mM to 75 μM, significantly increased tunw (Figure 1 – Figure Supplement 1f, [hexamer]=60 nM). The unwinding rate kunw, defined as the inverse of average tunw, vs. ATP concentration (red squares Figure 1 – Figure Supplement 1h) curve could be well-fitted using the Michaelis-Menten equation, yielding an apparent Michaelis-Menten constant Km = 87 ± 9 μM and a maximum unwinding rate kunw = 1.91 ± 0.05 s−1. Including ADP in the reaction increased the apparent Km to 191 ± 14 μM and 272 ± 36 μM at 0.5 mM and 1 mM ADP, respectively (Figure 1 – Figure Supplement 1g and h) with little change in the maximum unwinding rate kunw([ADP]=0.5mM) = 2.01 ± 0.04 s−1 and kunw([ADP]=1mM) = 1.91 ± 0.07 s−1. A fit to the more general Hill-equation to the ATP titration results in a Hill coefficient n=1.2 ± 0.2 (dashed line in Figure 1 – Figure Supplement 1h). A Hill coefficient of 1 implies that either each identical subunit can bind ATP and hydrolyze completely independent of each other (no binding cooperativity between the subunits) or that for every step one or more subunits can bind ATP successively and in coordination but not cooperatively (Schnitzer and Block, 1997). It is important to note that a Hill coefficient of 1 does not imply that only one ATP is bound per enzymatic cycle, but that binding of one ATP neither facilitates nor hinders binding of more ATP.
FRET calibration
FRET vs number of unwound base pairs was calibrated with the substrates 4AT4GC, 7AT33GC, 10AT30GC and 13AT27GC (Figure 1 – Figure Supplement 2c). All substrates allowed G40P only partial unwinding, e.g. the AT base pairs were unwound, followed by a stall at the GC base pairs. The stalling FRET level was determined through averaging over at least 5 data points at stalling events during an unwinding attempt (Figure 1 – Figure Supplement 2e through h). The FRET level distributions (Figure 1 – Figure Supplement 2d) were fitted with Gaussian distributions, yielding a FRET level of EFRET(4AT) = 0.73 ± 0.05 (± s), EFRET(7AT) = 0.61 ± 0.05, EFRET(10AT) = 0.48 ± 0.04, EFRET(13AT) = 0.24 ± 0.04 and a donor leakage of EFRET(leakage) = 0.11 ± 0.02. The GC base pair induced peaks fall within error on our intrinsic calibration (Figure 1 – Figure Supplement 2e) leading to the conclusion that G40P unwinds dsDNA in one base pair step size. Figure 1 – Figure Supplements 2e through h show example traces with 4AT, 7AT, 10AT and 13AT bp, respectively, followed by a GC bp stretch.
G40P ATPase activity and ATP and ATPγS affinity
ATPase activity of G40P was tested using EnzChek Phosphate Assay Kit (Invitrogen). The solution conditions were 20mM Tris-HCl, 50mM NaCl and either 10mM MgCl2 or 10mM MnCl2 and 1mM ATP. G40P showed without ssDNA a significant ATPase activity that could be increased after addition of ssDNA (Figure 3 – Figure Supplement 1a). This ATPase activity without ssDNA was suppressed with MnCl2 (Figure 3 – Figure Supplement 1b).
ATP and ATPγS affinity was determined using competitive titration as previously described by (Aregger and Klostermeier, 2009). We preformed complexes of G40P and Mant-ADP (Invitrogen) in 1:1 molar ratio (1μM G40P hexamer, 1μM mant-ADP). The final buffer conditions for the competitive titration was: 20mM Tris-HCl pH 8, 50mM NaCl, 10mM MnCl2. Mant-ADP was excited at 360 ± 5 nm, Mant emission was observed at 440 ± 5nm using Varian Cary Eclipse Fluorescence Spectrophotometer. The data was averaged over 5 seconds and 5 data points were taken and averaged. Addition of both, ATP or ATPγS, decreased the emission intensity indicating a competitive displacement of the prebound mant-ADP from G40P. The data was evaluated using a solution for a quadratic equation describing complex formation (Karow et al., 2007; Thrall et al., 1996) (Figure 3 – Figure Supplement 1c): where F0 denotes the unbound mant fluorescence, ΔFmax is the fluorescence amplitude, [Etot] the total enzyme concentration and [Ltot] the total ligand concentration and KD the apparent dissociation constant. Both ligands showed similar affinity (ATPγS KD= 1.4 ± 0.9 μM, ATP KD= 3.5 ± 0.8 μM).
Protein preparation
G40P and DnaG were expressed and purified as described previously (Wang et al., 2008).
Author contributions
M.S., X.S.C. and T.H. designed the research. G.W. expressed and purified the protein. M.S. performed the single-molecule experiments. M.S., G.W., X.S.C. and T.H. discussed the data. M.S. and T.H. analyzed the data and wrote the manuscript.
Competing interest
The authors declare no conflict of interest.
Acknowledgements
The authors thank Benjamin Leslie, Jaya Yodh, Prakrit Jena, Salman Syed, Jeehae Park, Sinan Arslan and Hannah Gelman for discussion. M.S. gratefully acknowledges his postdoctoral fellowship by German Research Foundation (DFG SCHL1896/1–1) and support by the German Ministry for Science and Education (BMBF 03Z2EN11). This work was supported by NIH grant GM065367 (to T.H.) and 137405 (to X.S.C.) and by NSF grant PHY-082261 (to T.H.).