Potent inhibition of DNA unwinding and ATPase activities of pea DNA helicase 45 by DNA-binding agents

Pea DNA helicase 45 (PDH45) is an ATP-dependent DNA unwinding enzyme, with intrinsic DNA-dependent ATPase activity [Plant J. 24 (2000) 219]. We have determined the effect of various DNA-binding agents, such as daunorubicin, ethidium bromide, ellipticine, cisplatin, nogalamycin, actinomycin C1, and camptothecin on the DNA unwinding and ATPase activities of the plant nuclear DNA helicase PDH45. The results show that all the agents except actinomycin C1, and camptothecin inhibited the helicase (apparent K(i) values ranging from 1.5 to 7.0 microM) and ATPase (apparent K(i) values ranging from 2.5 to 11.9 microM) activities. This is the first study to show the effect of various DNA-binding agents on the plant nuclear helicase and also first to demonstrate inhibition of any helicase by cisplatin. Another striking finding that the actinomycin C1 and ellipticine act differentially on PDH45 as compared to pea chloroplast helicase suggests that the mechanism of DNA unwinding could be different in nucleus and chloroplast. These results suggest that the intercalation of the inhibitors into duplex DNA generates a complex that impedes translocation of PDH45, resulting in both the inhibitions of unwinding activity and ATP hydrolysis. This study would be useful to obtain a better understanding of the mechanism of plant nuclear DNA helicase unwinding and the mechanism by which these agents can disturb genome integrity.     

The Gly-Arg-rich C-terminal domain of pea nucleolin is a DNA helicase that catalytically translocates in the 5'- to 3'-direction

Nucleolin is a major nucleolar phosphoprotein of exponentially growing eukaryotic cells. Here we report the cloning, purification, and characterization of the C-terminal glycine/arginine-rich (GAR) domain of pea nucleolin. The purified recombinant protein (17 kDa) shows ATP-/Mg(2+)-dependent DNA helicase and ssDNA-/Mg(2+)-dependent ATPase activities. The enzyme unwinds DNA in the 5'- to 3'-direction, which is the first report in plant for this directional activity. It unwinds forked/non-forked DNA with equal efficiency. The anti-nucleolin antibodies immunodepleted the activities of the enzyme. The DNA interacting ligands nogalamycin, daunorubicin, actinomycin C1, and ethidium bromide were inhibitory to DNA unwinding (with K(i) values of 0.40, 2.21, 8.0, and 9.0 microM, respectively) and ATPase (with K(i) values of 0.43, 1.65, 4.6, and 7.0 microM, respectively) activities of the enzyme. This study confirms that the unwinding and ATPase activities of pea nucleolin resided in the GAR domain. This study should make important contribution to our better understanding of DNA transaction in plants, mechanism of DNA unwinding, and the mechanism by which these ligands can disturb genome integrity.     

A novel nuclear DNA helicase with high specific activity from Pisum sativum catalytically translocates in the 3'-->5' direction.

A novel ATP-dependent nuclear DNA unwinding enzyme from pea has been purified to apparent homogeneity and characterized. This enzyme is present at extremely low abundance and has the highest specific activity among plant helicases. It is a heterodimer of 54 and 66 kDa polypeptides as determined by SDS/PAGE. On gel filtration chromatography and glycerol gradient centrifugation it gives a native molecular mass of 120 kDa and is named as pea DNA helicase 120 (PDH120). The enzyme can unwind 17-bp partial duplex substrates with equal efficiency whether or not they contain a fork. It translocates unidirectionally along the bound strand in the 3'-->5' direction. The enzyme also exhibits intrinsic single-stranded DNA- and Mg2+-dependent ATPase activity. ATP is the most favoured cofactor but other NTPs and dNTPs can also support the helicase activity with lower efficiency (ATP > GTP = dCTP > UTP > dTTP > CTP > dATP > dGTP) for which divalent cation (Mg2+ > Mn2+) is required. The DNA intercalating agents actinomycin C1, ethidium bromide, daunorubicin and nogalamycin inhibit the DNA unwinding activity of PDH120 with Ki values of 5.6, 5.2, 4.0 and 0.71 micro Ms, respectively. This inhibition might be due to the intercalation of the inhibitors into duplex DNA, which results in the formation of DNA-inhibitor complexes that impede the translocation of PDH120. Isolation of this new DNA helicase should make an important contribution to our better understanding of DNA transaction in plants.     

Inhibition of DNA helicase II unwinding and ATPase activities by DNA-interacting ligands. Kinetics and specificity.

Although DNA helicases play important roles in the processing of DNA, little is known about the effects of DNA-interacting ligands on these helicases. Therefore, the effects of a wide variety of DNA-binding ligands on the unwinding and ATPase reactions catalyzed by Escherichia coli DNA helicase II were examined. DNA minor groove binders and simple DNA intercalators did not inhibit helicase II. However, DNA intercalators, such as mitoxantrone and nogalamycin, which position functionalities in the major groove upon binding duplex DNA, were potent inhibitors of helicase II. To determine the mechanism by which mitoxantrone inhibited helicase II, the unwinding and DNA-dependent ATPase activities of helicase II were measured using a spectrum of double- and single-stranded DNA substrates. Using either a 71-base pair (bp) M13mp7 partially duplexed DNA substrate or a 245-bp bluntended, fully duplexed DNA substrate, the apparent Ki value for inhibition by mitoxantrone of both the unwinding and ATPase reactions was approximately 1 microM for both substrates, suggesting that the mechanism of inhibition of helicase II by mitoxantrone is the same for both substrates and requires the presence of double-stranded structure. To strengthen this conclusion, the ability of mitoxantrone to inhibit the DNA-dependent ATPase activity of helicase II was determined using two single-stranded substrates, poly(dT) and the 245-bp substrate after heat denaturation. Using either substrate, mitoxantrone inhibited the ATPase activity of helicase II far less effectively. Thus, these results indicate that the intercalation of mitoxantrone into double-stranded DNA, with accompanying placement of functionalities in the major groove, generates a complex that impedes helicase II, resulting in both inhibition of ATP hydrolysis and unwinding activity. Furthermore, we report here that DNA-binding ligands inhibit the unwinding activity of helicases I and IV and Rep protein from E. coli, demonstrating that the inhibition observed for helicase II is not unique to this enzyme.     

Characterization of replication fork and phosphorylation stimulated Plasmodium falciparum helicase 45

Helicases are essential enzymes, which play important role in the metabolism of nucleic acids. In the present study we report further characterization of PfH45 (Plasmodium falciparum helicase 45), which is an essential enzyme for parasite survival. The results show that the helicase activity of PfH45 is significantly stimulated by replication fork like structure. The studies using truncated derivatives of PfH45 show that its nucleic acid dependent ATPase activity resides in the N-terminal one third of the protein and its RNA and DNA-binding activity predominantly resides in the C-terminal two third of the protein. The phosphorylation of PfH45 by protein kinase C at Ser and Thr residues stimulated its DNA and RNA helicase and ssDNA and RNA-dependent ATPase activities. DNA-interacting compounds actinomycin, DAPI, daunorubicin, ethidium bromide, netropsin and nogalamycin were able to inhibit the helicase and ssDNA-dependent ATPase activity with apparent IC50 values ranging from 0.5 to 5.0 microM respectively. These compounds distinctively inhibit the helicase activity probably by forming complex with DNA and obstructing enzyme movement.     

Modulation of enzymatic activities of Escherichia coli DnaB helicase by single-stranded DNA-binding proteins

The modulation of enzymatic activities of Escherichia coli DnaB helicase by homologous and heterologous single-stranded DNA-binding proteins (SSBs) and its DNA substrates were analyzed. Although DnaB helicase can unwind a variety of DNA substrates possessing different fork-like structures, the rate of DNA unwinding was significantly diminished with substrates lacking a 3′ fork. A 5 nt fork appeared to be adequate to attain the maximum rate of DNA unwinding. Efficient helicase action of DnaB requires the participation of SSBs. Studies involving heterologous SSBs demonstrated that they can stimulate the helicase activity of DnaB protein under certain conditions. However, this stimulation occurs in a manner distinctly different from that observed with cognate E.coli SSB. The E.coli SSB was found to stimulate the helicase activity over a wide range of SSB concentrations and was unique in its strong inhibition of single-stranded DNA-dependent ATPase activity when uncoupled from the DNA helicase activity. In the presence of a helicase substrate, the ATPase activity of DnaB helicase remained uninhibited. Thus, E.coli SSB appears to coordinate and couple the ATPase activity to the DNA helicase activity by suppressing unproductive ATP hydrolysis by DnaB helicase.     

DNA binding and helicase actions of mouse MCM4/6/7 helicase

Helicases play central roles in initiation and elongation of DNA replication. We previously reported that helicase and ATPase activities of the mammalian Mcm4/6/7 complex are activated specifically by thymine-rich single-stranded DNA. Here, we examined its substrate preference and helicase actions using various synthetic DNAs. On a bubble substrate, Mcm4/6/7 makes symmetric dual contacts with the 5′-proximal 25 nt single-stranded segments adjacent to the branch points, presumably generating double hexamers. Loss of thymine residues from one single-strand results in significant decrease of unwinding efficacy, suggesting that concurrent bidirectional unwinding by a single double hexameric Mcm4/6/7 may play a role in efficient unwinding of the bubble. Mcm4/6/7 binds and unwinds various fork and extension structures carrying a single-stranded 3′-tail DNA. The extent of helicase activation depends on the sequence context of the 3′-tail, and the maximum level is achieved by DNA with 50% or more thymine content. Strand displacement by Mcm4/6/7 is inhibited, as the GC content of the duplex region increases. Replacement of cytosine–guanine pairs with cytosine–inosine pairs in the duplex restored unwinding, suggesting that mammalian Mcm4/6/7 helicase has difficulties in unwinding stably base-paired duplex. Taken together, these findings reveal important features on activation and substrate preference of the eukaryotic replicative helicase.     

An essential DnaB helicase of Bacillus anthracis: identification, characterization, and mechanism of action

We have described a novel essential replicative DNA helicase from Bacillus anthracis, the identification of its gene, and the elucidation of its enzymatic characteristics. Anthrax DnaB helicase (DnaB_BA) is a 453-amino-acid, 50-kDa polypeptide with ATPase and DNA helicase activities. DnaB_BA displayed distinct enzymatic and kinetic properties. DnaB_BA has low single-stranded DNA (ssDNA)-dependent ATPase activity but possesses a strong 5′→3′ DNA helicase activity. The stimulation of ATPase activity appeared to be a function of the length of the ssDNA template rather than of ssDNA binding alone. The highest specific activity was observed with M13mp19 ssDNA. The results presented here indicated that the ATPase activity of DnaB_BA was coupled to its migration on an ssDNA template rather than to DNA binding alone. It did not require nucleotide to bind ssDNA. DnaB_BA demonstrated a strong DNA helicase activity that required ATP or dATP. Therefore, DnaB_BA has an attenuated ATPase activity and a highly active DNA helicase activity. Based on the ratio of DNA helicase and ATPase activities, DnaB_BA is highly efficient in DNA unwinding and its coupling to ATP consumption.     

Structural and Mechanistic Insight into DNA Unwinding by Deinococcus radiodurans UvrD

DNA helicases are responsible for unwinding the duplex DNA, a key step in many biological processes. UvrD is a DNA helicase involved in several DNA repair pathways. We report here crystal structures of Deinococcus radiodurans UvrD (drUvrD) in complex with DNA in different nucleotide-free and bound states. These structures provide us with three distinct snapshots of drUvrD in action and for the first time trap a DNA helicase undergoing a large-scale spiral movement around duplexed DNA. Our structural data also improve our understanding of the molecular mechanisms that regulate DNA unwinding by Superfamily 1A (SF1A) helicases. Our biochemical data reveal that drUvrD is a DNA-stimulated ATPase, can translocate along ssDNA in the 3′-5′ direction and shows ATP-dependent 3′-5′, and surprisingly also, 5′-3′ helicase activity. Interestingly, we find that these translocase and helicase activities of drUvrD are modulated by the ssDNA binding protein. Analysis of drUvrD mutants indicate that the conserved β-hairpin structure of drUvrD that functions as a separation pin is critical for both drUvrD’s 3′-5′ and 5′-3′ helicase activities, whereas the GIG motif of drUvrD involved in binding to the DNA duplex is essential for the 5′-3′ helicase activity only. These special features of drUvrD may reflect its involvement in a wide range of DNA repair processes in vivo.     

Biochemical Characterization of Adeno-Associated Virus Rep68 DNA Helicase and ATPase Activities

The adeno-associated virus (AAV) nonstructural proteins Rep68 and Rep78 are site-specific DNA binding proteins, ATP-dependent site-specific endonucleases, helicases, and ATPases. These biochemical activities are required for viral DNA replication and control of viral gene expression. In this study, we characterized the biochemical properties of the helicase and ATPase activities of homogeneously pure Rep68. The enzyme exists as a monomer in solution at the concentrations used in this study (<380 nM), as judged by its mobility in sucrose density gradients. Using a primed single-stranded (ss) circular M13 substrate, the helicase activity had an optimum pH of 7 to 7.5, an optimum temperature of 45°C, and an optimal divalent-cation concentration of 5 mM MgCl₂. Several nucleoside triphosphates could serve as cofactors for Rep68 helicase activity, and the order of preference was ATP = GTP > CTP = dATP > UTP > dGTP. The K_m values for ATP in both the DNA helicase reaction and the site-specific trs endonuclease reaction were essentially the same, approximately 180 μM. Both reactions were sigmoidal with respect to ATP concentration, suggesting that a dimer or higher-order multimer of Rep68 is necessary for both DNA helicase activity and terminal resolution site (trs) nicking activity. Furthermore, when the enzyme itself was titrated in the trs endonuclease and ATPase reactions, both activities were second order with respect to enzyme concentration. This suggests that a dimer of Rep68 is the active form for both the ATPase and nicking activities. In contrast, DNA helicase activity was linear with respect to enzyme concentration. When bound to ssDNA, the enzyme unwound the DNA in the 3′-to-5′ direction. DNA unwinding occurred at a rate of approximately 345 bp per min per monomeric enzyme molecule. The ATP turnover rate was approximately 30 to 50 ATP molecules per min per enzyme molecule. Surprisingly, the presence of DNA was not required for ATPase activity. We estimated that Rep translocates processively for more than 1,300 bases before dissociating from its substrate in the absence of any accessory proteins. DNA helicase activity was not significantly stimulated by substrates that have the structure of a replication fork and contain either a 5′ or 3′ tail. Rep68 binds only to ssDNA, as judged by inhibition of the DNA helicase reaction with ss or double-stranded (ds) DNA. Consistent with this observation, no helicase activity was detected on blunt-ended ds oligonucleotide substrates unless they also contained an ss 3′ tail. However, if a blunt-ended ds oligonucleotide contained the 22-bp Rep binding element sequence, Rep68 was capable of unwinding the substrate. This means that Rep68 can function both as a conventional helicase for strand displacement synthesis and as a terminal-repeat-unwinding protein which catalyzes the conversion of a duplex end to a hairpin primer. Thus, the properties of the Rep DNA helicase activity suggest that Rep is involved in all three of the key steps in AAV DNA replication: terminal resolution, reinitiation, and strand displacement.     

Molecular and Functional Characterization of RecD,a Novel Member of the SF1 Family of Helicases,from Mycobacterium tuberculosis

The annotated whole-genome sequence of Mycobacterium tuberculosis revealed the presence of a putative recD gene; however, the biochemical characteristics of its encoded protein product (MtRecD) remain largely unknown. Here, we show that MtRecD exists in solution as a stable homodimer. Protein-DNA binding assays revealed that MtRecD binds efficiently to single-stranded DNA and linear duplexes containing 5′ overhangs relative to the 3′ overhangs but not to blunt-ended duplex. Furthermore, MtRecD bound more robustly to a variety of Y-shaped DNA structures having ≥18-nucleotide overhangs but not to a similar substrate containing 5-nucleotide overhangs. MtRecD formed more salt-tolerant complexes with Y-shaped structures compared with linear duplex having 3′ overhangs. The intrinsic ATPase activity of MtRecD was stimulated by single-stranded DNA. Site-specific mutagenesis of Lys-179 in motif I abolished the ATPase activity of MtRecD. Interestingly, although MtRecD-catalyzed unwinding showed a markedly higher preference for duplex substrates with 5′ overhangs, it could also catalyze significant unwinding of substrates containing 3′ overhangs. These results support the notion that MtRecD is a bipolar helicase with strong 5′ → 3′ and weak 3′ → 5′ unwinding activities. The extent of unwinding of Y-shaped DNA structures was ∼3-fold lower compared with duplexes with 5′ overhangs. Notably, direct interaction between MtRecD and its cognate RecA led to inhibition of DNA strand exchange promoted by RecA. Altogether, these studies provide the first detailed characterization of MtRecD and present important insights into the type of DNA structure the enzyme is likely to act upon during the processes of DNA repair or homologous recombination.     

Replication fork-stimulated eIF-4A from Plasmodium cynomolgi unwinds DNA in the 3' to 5' direction and is inhibited by DNA-interacting compounds

Plasmodium cynomolgi DEAD-box DNA helicase 45 (PcDDH45) is an ATP-dependent DNA-unwinding enzyme with intrinsic DNA-dependent ATPase activity and is highly homologous to eIF-4A. In this study, we have further characterized and tested the effect of various DNA-interacting compounds on the DNA-unwinding activity of PcDDH45. The results show that PcDDH45 translocates in the 3' to 5' direction along the bound strand, a replication fork-like structure of the substrate stimulates its DNA-unwinding activity, and it failed to unwind blunt-ended duplex DNA. Of various compounds tested, only cisplatin, 4',6'-diamidino-2-phenylindole, daunorubicin, and nogalamycin were inhibitory to the unwinding activity of PcDDH45 with apparent IC(50) values of 1.0, 4.0, 7.5, and 1.7 microM, respectively. These results suggest that the interaction of these compounds with duplex DNA generate a complex that probably impedes the translocation of PcDDH45, resulting in inhibition of unwinding activity. This study is one of the first to demonstrate the effect of various DNA-binding compounds on a malaria parasite DNA helicase and should make an important contribution to our better understanding of the nucleic acid transactions in the parasite.     

Plasmodium falciparum UvrD Helicase Translocates in 3′ to 5′ Direction,Colocalizes with MLH and Modulates Its Activity through Physical Interaction

Malaria is a global disease and a major health problem. The control of malaria is a daunting task due to the increasing drug resistance. Therefore, there is an urgent need to identify and characterize novel parasite specific drug targets. In the present study we report the biochemical characterization of parasite specific UvrD helicase from Plasmodium falciparum. The N-terminal fragment (PfUDN) containing UvrD helicase domain, which consists of helicase motifs Q, Ia–Id, II, III and most of motif IV, and the C-terminal fragment (PfUDC1) containing UvrD helicase C terminal domain, consisting of remaining part of motif IV and motifs IVa–IVc and 161 amino acids of intervening sequence between motif IV and V, possess ssDNA-dependent ATPase and DNA helicase activities in vitro. Using immunodepletion assays we show that the ATPase and helicase activities are attributable to PfUDN and PfUDC1 proteins. The helicase activity can utilize the hydrolysis of all the nucleotide and deoxynucleotide triphosphates and the direction of unwinding is 3′ to 5′. The endogenous P. falciparum UvrD contains the characteristic DNA helicase activity. PfUDN interacts with PfMLH (P. falciparum MutL homologue) and modulates the endonuclease activity of PfMLH and PfMLH positively regulates the unwinding activity of PfUDN. We show that PfUvrD is expressed in the nucleus distinctly in the schizont stages of the intraerythrocytic development of the parasite and it colocalizes with PfMLH. These studies will make an important contribution in understanding the nucleic acid transaction in the malaria parasite.     

A single subunit MCM6 from pea forms homohexamer and functions as DNA helicase

The initiation of DNA replication starts from origins and is controlled by a multiprotein complex, which involves about twenty protein factors. One of the important factors is hetrohexameric minichromosome maintenance (MCM2-7) protein complex which is evolutionary conserved and functions as essential replicative helicase for DNA replication. Here we report the isolation and characterization of a single subunit of pea MCM protein complex, the MCM6. The deduced amino acid (827) sequence contains all the known canonical MCM motifs including zinc finger, MCM specific Walker A and Walker B and arginine finger. The purified recombinant protein contains ATP-dependent 3′–5′ DNA helicase, ATP-binding and ATPase activities. The helicase activity was stimulated by replication fork like substrate and anti-MCM6 antibodies curtail all the enzyme activities of MCM6 protein. In vitro it self-interacts and forms a homohexamer which is active for DNA helicase and ATPase activities. The complete protein is required for self-interaction as the truncated MCM6 proteins were unable to self-interact. Western blot analysis and in vivo immunostaining followed by confocal microscopy showed the localization of MCM6 both in the nucleus and cytosol. These findings provide first direct evidence that single subunit MCM6 contains DNA helicase activity which is unique to plant MCM6 protein, as this activity was only reported for heteromultimers of MCM proteins in animal system. This discovery should make an important contribution to a better understanding of DNA replication in plants.     

Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding

Background

The mini-chromosome maintenance protein (MCM) complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex consists of six homologous proteins (MCM2-7), the archaeon Sulfolobus solfataricus has only one MCM protein (ssoMCM), six subunits of which form a homohexamer. We have recently reported a 4.35Å crystal structure of the near full-length ssoMCM. The structure reveals a total of four β-hairpins per subunit, three of which are located within the main channel or side channels of the ssoMCM hexamer model generated based on the symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM) structure. The fourth β-hairpin, however, is located on the exterior of the hexamer, near the exit of the putative side channels and next to the ATP binding pocket.

Results

In order to better understand this hairpin's role in DNA binding and helicase activity, we performed a detailed mutational and biochemical analysis of nine residues on this exterior β-hairpin (EXT-hp). We examined the activities of the mutants related to their helicase function, including hexamerization, ATPase, DNA binding and helicase activities. The assays showed that some of the residues on this EXT-hp play a role for DNA binding as well as for helicase activity.

Conclusions

These results implicate several current theories regarding helicase activity by this critical hexameric enzyme. As the data suggest that EXT-hp is involved in DNA binding, the results reported here imply that the EXT-hp located near the exterior exit of the side channels may play a role in contacting DNA substrate in a manner that affects DNA unwinding.

     

High-temperature single-molecule kinetic analysis of thermophilic archaeal MCM helicases

The minichromosome maintenance (MCM) complex is the replicative helicase responsible for unwinding DNA during archaeal and eukaryal genome replication. To mimic long helicase events in the cell, a high-temperature single-molecule assay was designed to quantitatively measure long-range DNA unwinding of individual DNA helicases from the archaeons Methanothermobacter thermautotrophicus (Mth) and Thermococcus sp. 9°N (9°N). Mth encodes a single MCM homolog while 9°N encodes three helicases. 9°N MCM3, the proposed replicative helicase, unwinds DNA at a faster rate compared to 9°N MCM2 and to Mth MCM. However, all three MCM proteins have similar processivities. The implications of these observations for DNA replication in archaea and the differences and similarities among helicases from different microorganisms are discussed. Development of the high-temperature single-molecule assay establishes a system to comprehensively study thermophilic replisomes and evolutionary links between archaeal, eukaryal, and bacterial replication systems.     

甲磺酸培氟沙星抑制大肠杆菌RecQ解旋酶的体外DNA结合、解链和ATPase活性

RecQ家族解旋酶是DNA解旋酶中高度保守的一个重要家族,参与DNA复制、修复、重组、转录及维持端粒稳定等细胞代谢过程,在维持染色体稳定性与完整性中起着重要作用．甲磺酸培氟沙星(pefloxacin mesylate,PFM)是一种新型氟喹诺酮类抗菌药物,对一些革兰氏阴性菌具有明显的杀菌效果,临床上已广泛使用．本研究利用荧光偏振、自由磷检测技术研究PFM对大肠杆菌RecQ解旋酶的DNA结合活性、解链活性、ATPase活性的影响．结果表明,低浓度PFM可促进大肠杆菌RecQ解旋酶与ssDNA、dsDNA结合,达到一定量后PFM则抑制酶与DNA底物的结合,这种影响与DNA底物有关;PFM对RecQ解旋酶的DNA解链活性和ATP酶活性都具有抑制作用,但其抑制的效果有极显著差异(P<0.01)：比较PFM对两种活性抑制的Ci值(对解链活性抑制的Ci值为(1.5±0.2) μmol/L,对ATP酶活性抑制的Ci值为(0.010±0.005) μmol/L)可知,PFM对大肠杆菌RecQ解旋酶ATPase活性的抑制强于其解链活性. 这些结果可为研究以DNA解旋酶为药物靶标的分子机理奠定相关理论基础．