Simultaneously monitoring DNA binding and helicase-catalyzed DNA unwinding by fluorescence polarization

Abstract

A new method for helicase-catalyzed DNA unwinding is described. This assay takes advantage of the substantial change in fluorescence polarization (FP) upon helicase binding and DNA unwinding. The low anisotropy value, due to the fast tumbling of the free oligonucleotide in solution, increases abruptly upon binding of helicase to the fluorescein-labeled oligonucleotide. The high anisotropy of the helicase– DNA complex decreases as the fluorescein-labeled oligonucleotide is released from the complex through helicase-catalyzed DNA unwinding. This FP signal can be measured in real time by fluorescent spectroscopy. This assay can simultaneously monitor DNA binding and helicase-catalyzed DNA unwinding. It can also be used to determine the polarity in DNA unwinding mediated by helicase. This FP assay should facilitate the study of the mechanism by which helicase unwinds duplex DNA, and also aid in screening for helicase inhibitors, which are of growing interest as potential anticancer agents.

INTRODUCTION

DNA and RNA helicases are a ubiquitous class of enzymes found in diverse species from Escherichia coli to human beings (1). The helicase-catalyzed unwinding of double-stranded nucleic acids is implicated in diverse nucleic acid metabolism processes (2). The unwinding of double-stranded DNA is required in processes such as replication, recombination and repair (3), while the unwinding of RNA duplexes is implicated in processes as diverse as transcription termination, translation initiation and RNA processing (4). Helicase translocates along the nucleic acid lattice in a unidirectional manner, while destabilizing the hydrogen bonds between the complementary base pairs by using the energy from the hydrolysis of nucleoside 5′-triphosphate, usually an ATP (2,3). Therefore, helicases are motor proteins that couple ATP hydrolysis to nucleic acid unwinding. Helicases display a polarity in DNA unwinding; some enzymes show a strong preference for unwinding duplex nucleic acids possessing a 3′ single-stranded DNA (ssDNA) flanking region, whereas others show a preference for a 5′ ssDNA flanking region. The discovery that several human genetic disorders such as Werner syndrome, Bloom syndrome, etc., have been linked to mutations in DNA helicases has greatly enhanced the interest in defining their mechanism of action (5,6).

A full description of the detailed mechanism by which helicase catalyzes duplex DNA unwinding requires a deeper understanding about the enzyme. The extensive kinetic studies of helicase-mediated DNA unwinding will provide valuable information towards elucidating the mechanism by which a helicase unwinds duplex DNA. For this reason, several continuous fluorometric assays in real time have been developed to measure the unwinding of duplex nucleic acids by helicase (7–11). In order to facilitate rapid kinetic studies, we have developed a new continuous helicase assay based on the fact that the free oligonucleotide tumbles rapidly with very rapid rotational diffusion and thus the fluorescein-labeled oligonucleotide has a low polarization. The helicase– oligonucleotide complex, having a much higher molecular weight, tumbles more slowly and the orientation of the polarized excitation remaining in the emission is higher: thus, the complex exhibits a higher polarization than the free-labeled oligonucleotide. Moreover, this higher fluorescence polarization (FP) signal will decrease as the fluorescent oligonucleotide is released from the DNA–helicase complex upon DNA unwinding. Therefore, the unwinding reaction can be followed by measuring the FP in real time. This assay facilitates kinetic studies and has the advantage that DNA binding and DNA unwinding can be observed in the same experiment. In addition, this method is quite sensitive and requires only minutes to be carried out.

MATERIALS AND METHODS

Reagents and buffers

All chemicals were reagent grade. All solutions were made with 18 MΩ (Milli-Ω plus) water. The temperatures and the concentrations of sodium chloride in the buffer are indicated throughout the text.

Oligonucleotides

The PAGE-purified unlabeled and fluorescein-labeled synthetic oligonucleotides were purchased from Genset (France). The structure and sequences of the DNA used in this study are shown in Table 1. Double-stranded oligonucleotides were obtained in a 20 mM Tris–HCl buffer (pH 7.2) containing 100 mM NaCl. The mixture was heated to 85°C for 5 min, and annealing was allowed by slow cooling to room temperature. The nomenclature of the two strands of the double-stranded DNA, defined as loading strand and non-loading strand as indicated in Figure 1, was used throughout the text.

Table 1. Oligonucleotide structures and sequences for DNA substrates.

Substrate	Duplex length (bp)	Structure and sequence
A	14	5′-AATCCGTCGAGCAGagttagggttagggttagggttag (t25)
		3′F-TTAGGCAGCTCGTC
B	22	5′-AATCCGTCGAGCAGAGTTAGGGttagggttagggttag (t25)
		3′F-TTAGGCAGCTCGTCTCAATCCC
C	14	5′F-AATCCGTCGAGCAGagttagggttagggttagggttag (t25)
		3′-TTAGGCAGCTCGTC
D	14	5′-AATCCGTCGAGCAGagttagggttagggttagggttag (t24)T-F
		3′-TTAGGCAGCTCGTC
E	36	5′-AATCCGTCGAGCAGAGTTAGGGTTAGGGTTAGGGTTag (t25)
		3′F-TTAGGCAGCTCGTCTGAATCCCAATCCCAATCCCAA
F	16	5′-AATCCGTCGAGCAGAGttagggttagggttagggttagctctagcagt (t18)
		3′F-TTAGGCAGCTCGTCTC
G	16	5′-aatccgtcgagcagagtta(t26)gttagGGTTAGCTCTAGCAGT
		CCAATCGAGATCGTCA-F-5′
H	40	5′-CAGCAGCGGGAATGTAACCATCGTTGGTCGGCAGCAGGGC(t25)
		3′F-GTCGTCGCCCTTACATTGGTAGCAACCAGCCGTCGTCCCG-5′

Figure 1.

(A) Schematic illustration of the FP-based unwinding assay. A fluorescein-labeled oligonucleotide (open circles) was annealed to the proximal end of a ssDNA molecule. This substrate alone gives a low anisotropy value. The anisotropy signal increases upon the binding of helicase (closed circles) to this DNA substrate. When the DNA strands are unwound by a helicase, the fluorescein-labeled oligo is released from the DNA–helicase complexes, and the anisotropy value is even lower than partial duplex DNA substrate. The unwinding reaction is followed by measurement of the anisotropy value. (B) DNA unwinding followed by fluorescence anisotropy. Fluorescein-labeled partial duplex DNA (5 nM) (black closed circles) was incubated in the unwinding buffer. The anisotropy value was increased and then decreased upon addition of 30 nM RecQ and 1 mM ATP, respectively.

Helicases

RecQ and UvrD helicases were purified from the overproducing E.coli strain under native conditions. Briefly, the bacteria were grown at 37°C in terrific broth supplemented with 50 µg/ml ampicillin. The expression of the protein was induced by adding isopropylthio-β-d-galactoside to 0.5 mM at low-log phase (OD₆₀₀ = 0.6). After 3 h of incubation, the cells were harvested and lysed. The lysate was centrifuged and applied to the column charged with histidine binding resin (Novagen). The protein bound to the column was eluted step-wise using 20 mM Tris–HCl (pH 7.9) buffer containing 100, 200, 300, 400 and 500 mM imidazole. The helicases containing fractions, identified by both DNA-dependent ATP hydrolysis and helicase activity assays, were pooled. The his-tag was cleaved using biotinylated thrombin during a dialysis step. Removal of biotinylated thrombin was accomplished using streptavidin–agarose magnetic beads (Novagen, Madison, WI). RecQ helicase was further purified by FPLC size-exclusion chromatography using a Superdex 200 column (Pharmacia). A lysine residue at amino acid position 55 (AAG) was mutated to alanine (GCG). The mutation was created using the QuikChange kit for Site-Directed Mutagenesis from Stratagene and confirmed by DNA sequencing (MWG Biotech). The mutant helicase RecQ(K55A) was expressed in E.coli BL21/DE3 cells and purified as described above for wild-type enzyme.

Unwinding assay

An unwinding assay was performed using a Beacon 2000 polarization instrument. An appropriate quantity of fluorescein-labeled duplex oligonucleotide was added to helicase unwinding buffer (150 µl total) in a temperature-controlled cuvette. The anisotropy was measured successively until it stabilized. The helicases were then added. When the higher anisotropy value became stable, the unwinding reaction was initiated by the rapid addition of ATP solution to give a final concentration of 1 mM. For the experiment performed with DNA trap, 56mer oligonucleotides were included in the ATP solution to give final concentrations of 1 mM and 2 µM for ATP and DNA trap, respectively. The decrease of the anisotropy was recorded every 8 s until it became stable. The unwinding buffer contained 25 mM Tris–HCl (pH 8), 30 mM sodium chloride, 3 mM magnesium acetate and 0.1 mM DTT.

RESULTS AND DISCUSSION

Rationale

Figure 1A shows a schematic representation of the one-strand fluorescently labeled DNA used to monitor helicase-catalyzed DNA unwinding. This assay is based on the observation that the FP of a free oligonucleotide is relatively low compared to that bound to the enzyme. A decrease in FP will be observed upon DNA unwinding when the fluorescein-labeled oligonucleotide is released from the helicase–DNA complex. As shown in Figure 1B, when DNA substrate A was incubated alone, the 3′ end fluorescein-labeled oligonucleotide gave a FP signal, and this signal was increased significantly upon addition of RecQ helicase, meaning the binding of the protein to the DNA substrate could be observed prior to DNA unwinding. The unwinding reaction was carried out by the addition of 1 mM ATP to the unwinding buffer containing the DNA–helicase complex at 25°C. The progress of the reaction was clearly followed by observing the decrease in FP, reflecting the unwinding and the release of the fluorescein-labeled ssDNA from the enzyme. From Figure 1B, one can note that the helicase-catalyzed DNA unwinding resulted in a decrease in FP even lower than partial duplex substrate alone. This value corresponds exactly to the value of protein-free fluorescein-labeled single-stranded oligonucleotide (data not shown), thus indicative of the complete unwinding of the DNA substrate.

Optimum conditions for observing helicase-mediated DNA unwinding

It is possible that the released fluorescein-labeled DNA strand could be rebound by free helicase in solution, and therefore no significant change of polarization value should be observed. In fact, we found that the rebinding of released fluorescein-labeled DNA largely depended on its fluorescein-labeled DNA length, free helicase concentration and ATP concentration. In accordance with the previous report (12), we have observed that RecQ helicase displays a high affinity only for ssDNA molecules longer than 20mer (X.G.Xi and H.Q.Xu, manuscript in preparation). As shown in Figure 2A, the released fluorescein-labeled DNA shorter than 20mer cannot rebind to RecQ helicase. Since many experiments were carried out using the fluorescein-labeled DNA which is shorter than 21mer, recycling of the released fluorescein-labeled oligonucleotide by RecQ helicase in solution does not occur even in the presence of a high concentration of enzyme.

Figure 2.

Effect of fluorescein-labeled DNA length protein and ATP concentration on the kinetics of duplex unwinding. (A) Kinetics of duplex unwinding with DNA formed with different lengths of fluorescein-labeled ssDNA as indicated. A 5 nM concentration of each DNA substrate and 40 nM RecQ protein were used in this study. (B) DNA substrate E (2 nM) (36 base duplex DNA) was incubated with 50 and 200 nM RecQ helicase. The DNA unwinding reaction was initiated by adding either 1 mM ATP (closed squares and open circles) or 1 mM ATP containing 2 µM ssDNA trap (closed circles). (C) DNA substrate A (2 nM) was incubated with 40 nM RecQ helicase. The unwinding reaction was initiated by the addition of different concentrations of ATP as indicated.

For the fluorescein-labeled DNA exceeding 21mer, its recycling by free RecQ helicase in solution depends on the concentration of enzyme (Fig. 2B). We reason that if an appropriate concentration of enzyme and large excess of ssDNA trap were used, there will be no significant amount of free enzyme in solution and the released fluorescein-labeled DNA should not be bound by helicase. As expected, Figure 2B shows that in the absence of ssDNA trap, the amplitude of DNA unwinding is ∼60% in the presence of 50 nM RecQ helicase, and it is further decreased to 30% as RecQ enzyme increased to 200 nM, suggesting a significant amount of the released 40mer fluorescein-labeled DNA was rebound by free RecQ protein. However, complete DNA unwinding can be observed in the presence of the ssDNA trap (Fig. 2B). Therefore, the anisotropy value corresponding to the protein-free fluorescein-labeled DNA can be observed. In this study, the concentrations of DNA and enzyme were chosen as 4–5 and 30–40 nM, respectively, since under these conditions, the amplitudes of DNA binding and DNA unwinding were maximum.

The recycling of the released fluorescein-labeled ssDNA depends also on the ATP concentration. Figure 2C shows that rebinding of the released loading strand to RecQ helicase is prevented as the ATP concentration increases. These results suggest that the binding of ATP to helicase may provoke a conformational change of RecQ helicase, which then possesses a lower affinity for ssDNA. Similarly, ATP-induced Rep monomer dissociation from DNA substrates has been observed (13).

Fluorescence polarization measures the RecQ helicase-catalyzed DNA unwinding

To further confirm that this assay does indeed measure the helicase-catalyzed DNA unwinding, we performed additional control experiments.

Unwinding is ATP dependent. It is well established that RecQ helicase requires ATP hydrolysis to promote DNA unwinding. AMPPNP and ATP-γ-S, the non-hydrolyzable analogs of ATP, inhibited the RecQ-mediated DNA unwinding (14). Figure 3A shows that the enzyme displays no detectable helicase activity either in the presence of 1 mM AMPPNP or ATP-γ-S. These results indicate that the observed decrease of FP arises from the unwinding of the DNA substrate by the helicase. In addition, corresponding well with the previous observations, dUTP, GTP, CTP and dTTP cannot be used by RecQ as the energy source to catalyze the DNA unwinding; their addition does not lead to a decrease of FP (Fig. 3A).

Figure 3.

(A) Effects of nucleotides on RecQ helicase-catalyzed DNA unwinding activity. DNA unwinding assays were performed as described in Materials and Methods using DNA substrate A (2 nM); each reaction contained 40 nM helicases and one of the listed nucleotides at a final concentration of 1 mM. The data presented are the average of three independent determinations. (B) Helicase-mediated DNA unwinding activity is ATP dependent. DNA substrate A (5 nM) was incubated with either 30 nM RecQ helicase or 30 nM ATPase-deficient mutant (K55A) enzyme and the unwinding reaction was initiated by addition of either 1 mM ATP (closed circles, wt helicase; open squares, mutant helicase) or 1 mM ATP containing 5 mM EDTA (closed squares).

The reaction is magnesium ion dependent. It has been shown that magnesium ions are required for the RecQ helicase activity (14). We demonstrated that in the absence of magnesium ions, RecQ helicases bind well to DNA substrate. To further show that the measured FP signal reflects the RecQ helicase unwinding activity, we performed two DNA unwinding experiments in which the helicases were first bound to labeled DNA. In one experiment, the reaction was initiated by the addition of 1 mM ATP, but the ATP sample also contained 5 mM EDTA, which should trap all magnesium ions in the reaction solution. As shown in Figure 3B (closed squares), a modest DNA unwinding was observed under this condition. In the other experiment, 5 mM EDTA was added to an ongoing helicase assay and the reaction was stopped immediately. However, this reaction could be restored by the addition of 10 mM magnesium acetate. Both experiments show that magnesium ions are critical to the DNA unwinding reaction.

The ATPase-deficient mutant RecQ (K55A) does not exhibit helicase activity. To further confirm that the observed FP signal reflects the helicase unwinding activity, a mutant of the RecQ helicase with impaired ATPase activity was created by site-directed mutagenesis. The lysine residue (K55), which is highly conserved in a nucleotide binding loop of the amino acid sequence (G/A)XXGXGK(T/S) (15), was substituted by an alanine residue. As expected, the mutant shows no detectable ATPase activity (results not shown). The ATPase-deficient mutant binds normally to DNA substrate; however, no unwinding activity was observed upon addition of ATP (Fig. 3B). Taken together, these experiments support the interpretation that the FP assay really does measure the helicase-catalyzed DNA unwinding.

RecQ remains associated with the loading strand after the completion of DNA unwinding

There are two possible interpretations of the observed decrease of FP upon the initiation by ATP. One case is that the decreased polarization accompanied the DNA unwinding as expected. The other is that helicase dissociates from the DNA–helicase complex upon addition of ATP, but without the DNA unwinding process. To rule out the latter possibility, the reaction was performed using the DNA substrates C and D which are identical to the substrate A that we used previously, except that the fluorescein molecule was attached to the 5′ or 3′ end of the loading strand DNA (substrates C and D) rather than to the 3′ end of the non-loading strand (see Fig. 1 for the definition of the nomenclature). Figure 4 shows that helicases bind normally to DNA substrates C and D as substrate A; however, no decrease of FP was observed upon addition of 1 mM ATP under the same conditions as in previous experiments. By carefully examining the curve, we detected a very small decrease in the fluorescence signal which should correspond to the dissociation of the unlabeled non-loading strand. This result suggested that RecQ helicase does not dissociate from the loading strand after hydrolysis of ATP; therefore, the latter possibility was eliminated. Thus, the decrease in FP that we measured using DNA substrate A really reflects the helicase-catalyzed DNA unwinding activity.

Figure 4.

RecQ helicase resides on the loading strand of DNA whereas the leading strand is excluded from the DNA–helicase complexes during the unwinding reaction. DNA substrates A, C, D (5 nM) were incubated with 30 nM RecQ helicase at 25°C and 1 mM ATP was then added. The anisotropy values were recorded every 8 s.

A very interesting discovery made with the FP assay is that RecQ helicase which possessed 3′-5′ polarity remains associated with the loading strand and excludes the non-loading strand after the completion of DNA unwinding. This observation also suggested that RecQ helicase translocates along the loading strand during unwinding. Whether helicase possessing 5′–3′ polarity remains on the loading strand or always remains on the non-loading strand during unwinding is not clear. Differentiation between these possibilities by the FP assay may be important for the elucidation of the molecular mechanism of polarity. This work is currently in progress in our laboratory.

Kinetic study of helicase by the FP assay

To assess whether the fluorescence depolarization method can be used to determine the kinetic parameters of helicase-catalyzed DNA unwinding, we performed single-turnover kinetics with respect to the DNA substrate. When DNA substrate A (14 base duplex DNA) was used, and in the absence of ssDNA trap, the reaction was first-order with a rate constant of 0.15 s^–1 (determined from Fig. 2), corresponding to an unwinding rate of 2.1 bases s^–1. When DNA substrate E (36 base duplex DNA) was used, the reaction amplitude (defined as the depolarization value of fluorescein-labeled ssDNA) decreased with increasing concentration of RecQ helicase as shown in Figure 2B. This phenomenon arises from the fact that the released fluorescein-labeled ssDNA can re-associate with high concentrations of non-DNA bound helicase. Therefore, a large excess of non-specific ‘trap’ DNA was added to prevent helicase from re-associating with released fluorescein-labeled ssDNA once it is released during the course of the reaction. In the presence of ssDNA trap, the amplitude arrived approached unity (Fig. 2B).

To determine whether the observed rate constant varies as a function of the length of the duplex DNA, we performed single-turnover kinetics with three DNA substrates (substrates A, B and E) containing 14, 22 and 40 base duplex DNA (Fig. 5). The results show that the apparent rate constant decreased with the longer length of duplex DNA. This suggests that the observed rate constant truly reflects kinetic steps linked with duplex DNA unwinding. As shown in Table 2, the apparent duplex DNA unwinding rate varied from 2.8 to 1.3 bp s^–1 for 14, 22 and 40 base duplex DNA. The average of the unwinding rate is ∼2 bp s^–1, which is consistent with the previous observation (12).

Figure 5.

The effect of duplex DNA length on the single-turnover kinetics of RecQ-mediated DNA unwinding. RecQ helicase (50 nM) was incubated with 2 nM DNA substrtate A (circles, 14 bases in duplex length), substrate B (squares, 22 bases in duplex length), substrate E (rhombus, 40 bases in duplex length); DNA unwinding was initiated upon addition of 1 mM ATP containing 2 µM ssDNA trap at 25°C. Data from all three time courses were fitted to the exponential equation: A_t = A exp(–k_obst), where A_t is the anisotropy amplitude at time t, and k_obs is the observed rate constant. The insert shows the fraction unwound of DNA substrates.

Table 2. Rate constants determined with different DNA substrates.

DNA substrate	Duplex length	kobs	R2	Unwinding rate
A	14	0.202 ± 0.0025	0.998	2.8
B	22	0.055 ± 0.0015	0.964	1.4
E	40	0.032 ± 0.0004	0.993	1.3

The observed rate constants were determined from the fits of Figure 5.

Effect of temperature on unwinding as measured by the FP assay

The observed rate constants, k_obs, at different temperatures measured by the FP assay, were shown plotted logarithmically against 1/T in Figure 6. The apparent energy of activation (E_a) can be determined. Using data from reactions performed at temperatures between 4 and 37°C, Arrhenius plots were generated (Fig. 6). The slopes of these plots yield E_a values of 4.1 and 5.7 kcal/mol for 14 and 22 bp DNA, respectively. These values are compatible with those obtained from other types of assay, showing that the FP assay is a reliable method for measuring DNA unwinding activity.

Figure 6.

Arrhenius plots of the observed unwinding rate of RecQ helicase. The experiments were performed under standard conditions at temperatures between 4 and 37°C. DNA substrates A and B (2 nM) and 40 nM RecQ helicase were used.

Helicase polarity determination by the FP assay

DNA molecules display an intrinsic asymmetry due to the complementary double helix on opposite strands. Therefore, helicases generally show a distinct polarity in their DNA unwinding reaction (2). It seems that this polarity was determined by the presence of a ssDNA region flanking the duplex (2,16). Typically, a linear ssDNA annealed with radiolabeled complementary oligonucleotides at both ends was incubated with helicase and the polarity of unwinding was monitored by native gel electrophoresis and autoradiography (17). Depending on whether the 3′ or 5′ DNA end was released, the polarity of unwinding is referred to as 3′ to 5′ or 5′ to 3′, respectively. However, this assay requires extensive manipulation and is very time consuming. Using the FP assay, the same task can be accomplished in a few minutes. In this experiment, two substrates were used: one consisting of a linear ssDNA hybridized at its 5′ end with fluorescence oligonucleotide (substrate F). The other was essentially the same except that a fluorescent oligonucleotide at its 3′ end was hybridized with the linear ssDNA (substrate G). As shown in Figure 7, when the substrate F was used, RecQ helicase-mediated DNA unwinding activity was observed upon addition of ATP, whereas when the substrate G was used, no activity or modest activity was observed, clearly demonstrating that RecQ helicase possesses a 3′–5′ polarity. Similar results were obtained with UvrD helicase. Thus, the FP assay can be used to determine the polarity displayed by helicase. In addition, for the helicase which initiates only at blunt double-stranded DNA, such as RecBC helicase, it was impossible to determine the polarity using the conventional gel electrophoresis method until the various gapped DNA substrates were used (18). If an appropriate fluorescein-labeled DNA substrate was used, the FP assay developed in this report is an alternative method to determine the polarity of the helicase which initiates only at blunt double-stranded DNA.

Figure 7.

Helicase polarity determination by fluorescence anisotropy assay. RecQ helicase (30 nM) or UvrD helicase (30 nM) was incubated, respectively, with 2 nM partial duplex DNA substrate F or G, and DNA unwinding was initiated by the addition of 1 mM ATP.

In summary, we have developed a reliable, rapid, continuous, real time helicase assay. DNA binding and helicase-catalyzed DNA unwinding can be simultaneously monitored. The polarity of the enzyme, the identity of the helicase associated strand and translocation during unwinding can all be determined with this method. In addition, this method can also be used to study the RNA binding and RNA helicase-catalyzed unwinding activity.