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Acta Biochim Biophys Sin 2005,37:593-600
doi:10.1111/j.1745-7270.2005.00084.x

RecQ Helicase-catalyzed DNA Unwinding Detected by Fluorescence Resonance Energy Transfer

 

Xing-Dong ZHANG, Shuo-Xing DOU, Ping XIE, Peng-Ye WANG*, and Xu Guang XI1

 

Laboratory of Soft Matter Physics, Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100080, China;

1 Laboratoire de Biotechnologies et Pharmacologie Généique Appliquée CNRS UMR 8113, Ecole Normale Supérieure de Cachan, 61 Avenue du Président Wilson, 94235 Cachan cedex, France

 

Received: March 17, 2005

Accepted: May 23, 2005

This work was supported by the grants from the National Natural Science Foundation of China (No. 60025516 and No. 10334100), the Innovation Project of the Chinese Academy of Sciences, and the Centre­ National de la Recherche Scientifique (CNRS)

Abbreviations: FRET, fluorescence resonance energy transfer; H, hexachlorofluorescein; F, fluorescein; dsDNA, double-stranded DNA; ssDNA, single-stranded DNA; DTT, dithiothreitol

*Corresponding author: Tel, 86-10-82649568; Fax, 86-10-82640224; E-mail, [email protected]

 

Abstract        A fluorometric assay was used to study the DNA unwinding kinetics induced by Escherichia coli RecQ helicase. This assay was based on fluorescence resonance energy transfer and carried out on stopped-flow, in which DNA unwinding was monitored by fluorescence emission enhancement of fluorescein­ resulting from helicase-catalyzed DNA unwinding. By this method, we determined the DNA unwinding rate of RecQ at different enzyme concentrations. We also studied the dependences of DNA unwinding magnitude and rate on magnesium ion concentration. We showed that this method could be used to determine the polarity of DNA unwinding. This assay should greatly facilitate further study of the mechanism for RecQ-catalyzed DNA unwinding.

 

Key words        helicase; DNA unwinding; kinetics; stopped-flow; fluorescence resonance energy transfer (FRET)

 

DNA and RNA helicases are unwinding enzymes that can couple nucleotide triphosphate (usually ATP) binding and hydrolysis to catalyze breakage of the hydrogen bonds between complementary strands of double-stranded DNA (dsDNA), thus providing single-stranded DNA (ssDNA) templates required for almost all aspects of nucleic acid metabolic pathways [16]. These enzymes seem to be ubiquitous in both prokaryotic and eukaryotic cells as they have been found in diverse species from bacteria to human [7-13]. It has been reported widely, since this type of enzyme was originally described and recognized by Hoffmann-Berling in Escherichia coli almost 30 years ago, that DNA helicases play essential roles in a variety of biological processes such as DNA replication, repair, recombination and transcription [14,15], and defects in DNA helicases can cause genomic instability. It has been shown that one class of DNA helicases, the RecQ family, plays an important role in maintenance of genome stability [1618]. We now know that several human diseases such as Bloom's syndrome, Werner's syndrome and Rothmund-Thomson syndrome, all of which can lead to cancer, are caused by defection of RecQ helicase [1922]. The RecQ family helicases BLM and WRN are mutated in Bloom's syndrome and Werner's syndrome, respectively, and RecQ4 is defective in Rothmund-Thomson syndrome. Recently, the DNA unwinding mechanism of RecQ helicases has gained wide interest owing to their role in inherited diseases, as well as in aging.

To better understand how helicases unwind the duplex nucleic acids, many continuous fluorometric assays have been developed to study their detailed kinetic unwinding mechanisms. Up to now, various types of helicases have been studied by fluorescence methods, such as PcrA [23], RecBCD [24,25], Dda [26,27], Rep [28,29], Werner's syndrome protein as well as RecQ [30,31]. Recently, we have used a fluorescence polarization assay in the biochemical characterization of RecQ helicase and this method provided much valuable kinetic information for elucidating the unwinding mechanism of E. coli RecQ in real time [3234]. In particular, this fluorescence assay can simultaneously monitor DNA binding and helicase-catalyzed DNA unwinding.

Fluorescence resonance energy transfer (FRET) assay is an adaptation of similar spectrophotometric experiments performed with several other helicases such as WRN and Rep [28,30]. In this method, the dsDNA substrate was typically labeled with fluorescein (F, donor) at the 3' end and with hexachlorofluorescein (H, acceptor) at the opposing­ 5' end. The overlap between the fluorescence emission spectrum of fluorescein and the excitation spectrum­ of hexachlorofluorescein results in an energy transfer from fluorescein to hexachlorofluorescein, which quenches the fluorescein emission in the duplex. If the dsDNA is unwound or the complementary strands are separated­ by helicases, fluorescein and hexachloro­fluorescein are no longer in close proximity so the energy transfer is disrupted, resulting in an enhancement of fluorescence­ emission from the fluorescein. One of the advantages of this assay is that it can non-invasively measure­ the real-time kinetics in a millisecond time range and allow DNA unwinding activity to be monitored continuously­ at DNA concentrations as low as 2 nM. By the FRET method, we have determined the DNA unwinding­ rate of RecQ at different enzyme concentrations under multi-turnover conditions. The observed unwinding rate at saturating enzyme concentration is about 3 bp/s, which is consistent with that measured by using fluorescence polarization assay [32]. We also studied the dependences of both unwinding magnitude and unwinding rate on magnesium­ ion concentration and obtained results that are significantly­ different from those reported previously [31].

In this study, we used the stopped-flow rapid chemical kinetics method to observe the RecQ-catalyzed unwinding­ of dsDNA continuously and in real time based on FRET assay.

 

 

Materials and Methods

 

 

Reagents and buffers

 

All chemicals were of reagent grade and all solutions were prepared in high quality de-ionized water from a Milli-Q water purification system (Millipore Corporation, France) that has a resistivity greater than 18.2 MW∙m. All unwinding reactions were performed in buffer A [25 mM Tris-HCl, pH 7.4, 50 mM NaCl, 3 mM MgCl2 and 0.1 mM dithiothreitol (DTT)] at 25 ºC, unless otherwise mentioned in the text. ATP was purchased from Sigma (St. Louis, USA) and was dissolved as a concentrated stock at pH 7.0. The ATP concentration was determined at 259 nM using an extinction coefficient of 1.54´10⁴ cm^-1∙M^-1. The temperature and concentration of salts in other solutions­ are indicated throughout this paper.

 

RecQ protein preparation

 

His6-tagged E. coli RecQ helicase was expressed from pET-15b expression plasmid in E. coli strain BL21(DE3) as previously described [33] and was quantified spectrophotometrically at 280 nm using an extinction coefficient of 3.0´10⁴ cm^-1∙M ^-1. The purified E. coli RecQ protein was stored in buffer B (25 mM Tris-HCl, pH 7.4, 50 mM NaCl, 3 mM MgCl2 and 2 mM DTT) at -80 ºC. The purity of the protein was analyzed by Coomassie blue stained SDS-PAGE and electrospray mass spectrometry. Mutant RecQ(K55A) protein was prepared 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), was substituted by an alanine residue as previously described [32].

 

Oligonucleotide reaction substrates

 

All oligonucleotide types used in this research are listed in Table 1. Single-stranded oligonucleotides, with or without­ fluorescent­ label, were purchased from SBS GeneTech Company, Limited (Beijing, China), and all synthetic­ oligonucleotides­ were purified by denaturing polyacrylamide­ gel electrophoresis before storage in 10 mM Tris-HCl (pH 8.0) containing 1 mM EDTA at -20 ºC. A 50 mM stock solution of dsDNA was prepared by mixing­ the same amount of complementary single-stranded oligonucleotides­ in a 20 mM Tris-HCl buffer (pH 7.4 at 25 ºC) containing 100 mM NaCl, followed by heating to 85 ºC. After equilibrating for 5 min, annealing was allowed­ by slowly cooling off to room temperature. Duplexes were stored at -20 ºC.

 

Steady-state fluorescence spectrum measurement

 

Steady-state fluorescence spectra were measured using­ an F-4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). The samples were incubated in unwinding­ reaction buffer in a 1 cm square quartz cuvette with a magnetic stirring bar and the measurements were performed­ at 25 ºC. The concentration of oligonucleotides used in each experiment is specified in the figure legends. Fluorescence spectra were corrected by subtraction of the solvent spectra measured under the same conditions.

 

Stopped-flow fluorescence measurement

 

The stopped-flow fluorescence experiments were carried­ out using a Bio-Logic SFM-400 mixer with a 1.5 mm´1.5 mm cell (Bio-Logic FC-15) and a Bio-Logic MOS450/AF-CD optical system equipped with a 150 W mercury-xenon lamp. Fluorescein was excited at 492 nm (2 nm in slit width) and the fluorescence emission­ was monitored using a 525 nm high pass filter (Chroma Technology Company). RecQ and dsDNA substrates were pre-incubated­ at 25 ºC in a large syringe (syringe 1, 10 ml volume) for 5 min; ATP was in a small syringe (syringe 4, 1.9 ml volume). Each syringe contained unwinding reaction­ buffer A. In this experiment, the mixing ratio of syringe 1 versus syringe 4 was designed as 4:1. That is, concentrations of RecQ and dsDNA substrates in syringe 1 were 1.25-fold of the indicated final concentrations; the concentration of ATP in syringe 4 was diluted 5-fold when the unwinding reaction was initiated by mixing. The final concentrations of DNA, RecQ and ATP after mixing can be calculated from the mixing ratio. To convert the output­ fluorescence signal in volts to the fraction unwound, we performed another experiment in four-syringe mode, where RecQ protein in syringe 1, hexachlorofluorescein-labeled single-stranded oligonucleotides in syringe 2, and fluorescein-labeled single-stranded oligonucleotides in syringe 3 were incubated in unwinding reaction buffer, and the solution in syringe 4 was the same as in the above unwinding experiment. The fluorescent signal of the mixed solution from the four syringes corresponded to 100% unwinding. All kinetic traces shown in this paper were analyzed using software supplied by Bio-Logic (Bio-Kine32, version 4.26). All the solutions were filtered and extensively degassed immediately before they were used. The stopped-flow temperature was controlled by means of an external thermo-stated water bath and a high flux pump to circulate the water between the bath and the stopped-flow apparatus. Standard reaction temperature was 25 ºC.

 

Kinetics data analysis

 

The kinetics data of DNA unwinding were fitted to Equation­ 1.

Eq. 1

in which, F(t) is the fluorescence signal given in the fraction­ of duplex unwound; A and kobs correspond to the amplitude­ and the observed rate constant of DNA unwinding, respectively, t is the DNA unwinding time.

 

 

Results and Discussion

 

 

Purity of the RecQ helicase

 

The RecQ helicase was overexpressed and purified from E. coli [33]. Gel electrophoresis of the protein in denaturing­ conditions gave a single band corresponding to a molecular­ mass of about 70 kDa (Fig. 1). This is consistent with the value determined from the amino acid sequence (68,290 Da). The purity and the molecular mass of the protein were further confirmed by electrospray mass spectrometry, which shows that the purity exceeds 95%.

 

Monitoring dsDNA unwinding with FRET

 

The principle of the method is schematically shown in Fig. 2. The upper strand of the duplex was labeled with hexachlorofluorescein at the 5' end, whereas the complementary strand was labeled with fluorescein at the 3' end. The fluorescence emission spectrum of 3'-fluorescein-labeled­ ssDNA and the excitation and emission spectra of 5'-hexachlorofluorescein-labeled ssDNA are shown in Fig. 3(A). It can be seen there is a large spectrum overlap between the fluorescein emission and hexachlorofluorescein excitation spectra. After annealing and formation of dsDNA, when the fluorescein and hexachlorofluorescein are in close proximity, FRET will occur between the two fluorescent molecules. Fluorescence emission of fluorescein (525 nm) becomes reduced and that of hexachlorofluorescein (556 nm) enhanced [Fig. 3(B)]. In the case of dsDNA unwinding studied, the situation is reversed, that is, fluorescence emission of fluorescein (525 nm) becomes enhanced and that of hexachlorofluorescein (556 nm) reduced.

To determine whether the enhancement of fluorescein emission at 525 nm will be detected as a result of dsDNA unwinding by RecQ, we performed fluorescence measurement under unwinding reaction conditions before and after initiation of the unwinding activity. RecQ protein was pre-incubated with fluorescent-labeled dsDNA substrate in unwinding buffer at 25 ºC, and the emission spectrum was measured. After the addition of ATP, the emission spectrum measurement was performed (Fig. 4). The observed enhancement of fluorescein emission at 525 nm is consistent with a loss of FRET after the dsDNA substrate was unwound by RecQ.

 

Kinetics of RecQ-catalyzed DNA unwinding

 

Using the stopped-flow fluorescence technique, we studied the kinetic process of RecQ-catalyzed dsDNA unwinding by monitoring the fluorescence emission enhancement of fluorescein at 525 nm. We performed the experiments at a fixed dsDNA concentration and varying helicase concentration. The dsDNA substrate and RecQ helicase were mixed and incubated at 25 ºC for 5 min in unwinding buffer A in syringe 1 and the unwinding reaction was initiated by rapid mixing with ATP from syringe 4. The results are given in Fig. 5, where the concentration of fluorescein-labeled dsDNA is fixed at 2 nM and the kinetic trace at each RecQ concentration was obtained from an average of three measurements. The unwinding amplitude (the fraction of duplex unwound) and the observed rate constant were determined by fitting the experimental results with Equation 1. They are summarized in Table 2 and shown in Fig. 6. It can be seen that the unwinding amplitude and unwinding rate increase with increasing [RecQ]. The unwinding amplitude starts to saturate at [RecQ]=40 nM while the unwinding rate starts to saturate at slightly higher [RecQ], indicating that dsDNA substrates are completely saturated by RecQ molecules. The maximum unwinding rate is approximately 3 bp/s, which is consistent with previous experimental results [32].

 

Effect of Mg2+ and ATP on unwinding activity

 

We studied the effect of Mg2+ and ATP on RecQ helicase activity. It has been shown that magnesium ions are required­ for the helicase activity of RecQ and other helicases [31,35,36]. It was reported that the helicase activity­ of RecQ is unexpectedly optimal at a free Mg2+ concentration of 0.05 mM [31]. Here we performed kinetic­ DNA unwinding experiments with different concentrations of Mg2+ with typical results given in Fig. 7. We observed that no unwinding was observed when RecQ helicase and dsDNA substrate were incubated in the unwinding buffer without Mg2+, whereas unwinding was observed when the unwinding reaction was stimulated by adding MgCl2. At low Mg2+ concentrations (<1 mM), the initial unwinding­ rate of RecQ helicase increases with the concentration of Mg2+. At higher concentrations of Mg2+ (1 mM to 10 mM), it does not change significantly. When the concentration of Mg2+ reaches 50 mM, the unwinding rate of RecQ helicase becomes very small and the amplitude of unwinding decreases significantly. This experiment demonstrated that magnesium ions are important for the DNA unwinding reaction. The significant difference between these results and those already published may be due to the fact that long dsDNA (>2 kb) and single-stranded DNA-binding protein have been used previously [31].

It should be noted that in our previous work [32], a residual DNA unwinding activity was observed without magnesium, but no background unwinding was observed in our present experiments. One possible reason is that the trapping of Mg2+ by EDTA disturbs the stability of DNA-RecQ complexes and leads to a decrease of the measured anisotropy [32]. In the present experiments, there would be no change of fluorescence signal as long as the two strands of dsDNA are not separated.

It is well established that ATP hydrolysis is required for dsDNA unwinding by RecQ helicase [32,35]. As shown in Fig. 8, DNA unwinding was not observed before the addition of ATP to initiate the unwinding reaction by RecQ helicase. As a comparison, we performed another experiment that the unwinding reaction was triggered by the addition of 1 mM ATP in syringe 4. This experiment showed that the RecQ helicase has robust unwinding activity in the presence of ATP. Thus the unwinding reaction catalyzed by RecQ is completely ATP dependent.

Using the ATPase-deficient mutant, RecQ(K55A) [32], we did not observe any fluorescence enhancement at 525 nm under the condition when wild-type (WT) RecQ helicase unwinds DNA, demonstrating that the ATPase-deficient mutant RecQ (K55A) is incapable of catalyzing dsDNA unwinding (Fig. 8).

 

Polarity of DNA unwinding by RecQ

 

Because DNA helicases are involved in the initiation of replication [5], they may display specific polarity when they translocate on either the leading (3'®5' polarity) or lagging (5'®3' polarity) strand. It is now generally believed­ that the observed polarity requirement of helicases is a consequence of a directional bias in translocation on the ssDNA template (i.e. 3'®5' or 5'®3'). The polarity of unwinding by DNA helicases can usually be determined using a DNA substrate consisting of a linear ssDNA template­ flanking the duplex region at one end. Similar to many other helicases [37], RecQ requires an ssDNA overhanging­ adjacent to the duplex for initiating unwinding­ activity. We have used fluorescence polarization assay to determine that the unwinding polarity of RecQ is 3'®5'. Native gel electrophoresis and autoradiography helicase assay also gave the same results [35,36]. In our present study, we used two types of dsDNA substrates: one containing­ a 3'-ssDNA tail (substrate I) and the other containing­ a 5'-ssDNA tail (substrate III). In accordance with previous studies, dsDNA with a 5'-ssDNA tail is not recognized or unwound by RecQ (Fig. 9). Thus RecQ has a DNA unwinding activity characteristic of a DNA helicase with a 3'®5' polarity. We also used a blunt-ended dsDNA (substrate II) to detect the helicase activity of RecQ. No fluorescence enhancement was detected after addition of ATP as the concentration of RecQ reached 50 nM (data not shown). This assay could be widely applied to the determination of unwinding polarity of different helicases, regardless of monopolar or bipolar helicases, provided that a suitable, non-inhibitory fluorescent dye is selected to label duplex DNA substrates [26-28].

 

Conclusion

 

The fluorometric stopped-flow assay based on FRET has been proven to be a powerful method for the study of DNA unwinding characteristics of helicases such as Rep protein and WRN helicase [28,30]. Similar fluorescence assays have been widely used to study other helicases [38-40]. In this work, we showed that this stopped-flow FRET assay gave some results which are consistent with those obtained from other assays [31,32,35]. To study the kinetic mechanisms of DNA unwinding by RecQ helicase, this stopped-flow FRET assay is ideal because it can provide continuous kinetics data in the millisecond to second time range. In addition, as demonstrated in the present work, this fluorescence assay is extremely sensitive, allowing DNA unwinding to be monitored continuously and in real time at dsDNA concentrations as low as 2 nM. This assay can be further used to study the rapid kinetic mechanism by which RecQ helicase unwinds duplex DNA. For example, it is known that RecQ functions as a monomer [33], but is there any functional interaction of RecQ molecules during DNA unwinding, as in the case of hepatitis C virus helicase [41]? Combined with traditional electrophoresis assay and single-molecular helicase assay [33,42], this fluorometric stopped-flow assay may enable us to find answers to such important questions.

 

 

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