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.     

A maltose-binding protein/adeno-associated virus Rep68 fusion protein has DNA-RNA helicase and ATPase activities.

The adeno-associated virus type 2 (AAV) Rep68 protein produced in Escherichia coli as a fusion protein with maltose-binding protein (MBP-Rep68 delta) has previously been shown to possess DNA-DNA helicase activity, as does the purified wild-type Rep68. In the present study, we demonstrate that MBP-Rep68 delta also catalyzes the unwinding of a DNA-RNA hybrid. MBP-Rep68 delta-mediated DNA-RNA helicase activity required ATP hydrolysis and the presence of Mg2+ ions and was inhibited by high ionic strength. The efficiency of the DNA-RNA helicase activity of MBP-Rep68 delta was comparable to its DNA-DNA helicase activity. However, MBP-Rep68 delta lacked the ability to unwind a blunt-ended DNA-RNA substrate and RNA-RNA duplexes. We have also demonstrated that MBP-Rep68 delta has ATPase activity which is enhanced by the presence of single-stranded DNA but not by RNA. The MBP-Rep68 delta NTP mutant protein, which has a lysine-to-histidine substitution at amino acid 340 in the putative nucleoside triphosphate-binding site of Rep68, not only lacks DNA-RNA helicase and ATPase activities but also inhibits the helicase activity of MBP-Rep68 delta. DNA-RNA helicase activity of Rep proteins might play a pivotal role in the regulation of AAV gene expression by AAV Rep proteins.     

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．     

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.     

Biochemical and kinetic characterization of the DNA helicase and exonuclease activities of werner syndrome protein

The WRN gene, defective in the premature aging and genome instability disorder Werner syndrome, encodes a protein with DNA helicase and exonuclease activities. In this report, cofactor requirements for WRN catalytic activities were examined. WRN helicase performed optimally at an equimolar concentration (1 mm) of Mg(2+) and ATP with a K(m) of 140 microm for the ATP-Mg(2+) complex. The initial rate of WRN helicase activity displayed a hyperbolic dependence on ATP-Mg(2+) concentration. Mn(2+) and Ni(2+) substituted for Mg(2+) as a cofactor for WRN helicase, whereas Fe(2+) or Cu(2+) (10 microm) profoundly inhibited WRN unwinding in the presence of Mg(2+).Zn(2+) (100 microm) was preferred over Mg(2+) as a metal cofactor for WRN exonuclease activity and acts as a molecular switch, converting WRN from a helicase to an exonuclease. Zn(2+) strongly stimulated the exonuclease activity of a WRN exonuclease domain fragment, suggesting a Zn(2+) binding site in the WRN exonuclease domain. A fluorometric assay was used to study WRN helicase kinetics. The initial rate of unwinding increased with WRN concentration, indicating that excess enzyme over DNA substrate improved the ability of WRN to unwind the DNA substrate. Under presteady state conditions, the burst amplitude revealed a 1:1 ratio between WRN and DNA substrate, suggesting an active monomeric form of the helicase. These are the first reported kinetic parameters of a human RecQ unwinding reaction based on real time measurements, and they provide mechanistic insights into WRN-catalyzed DNA unwinding.     

A DNA helicase from Xenopus laevis ovaries

A DNA helicase was extensively purified from Xenopus laevis ovaries. The most purified fraction was free of DNA topoisomerase, DNA polymerase, and nuclease activities. The enzyme had a Stokes radius of 54 A and a sedimentation coefficient of 6-7.3 S, from which a native molecular weight of 140,000-170,000 was calculated. DNA helicase activity required Mg2+ or Mn2+ and was dependent on hydrolysis of ATP or dATP. Monovalent cations, K+ and Na+, stimulated DNA unwinding with an optimum at 130 mM. DNA-dependent ATPase activity copurified with the X. laevis DNA helicase. Double-stranded and single-stranded DNA were both cofactors for the ATPase activity, but single-stranded DNA was more efficient. The molecular weight, monovalent cation dependence, cofactor requirements, and elution from single-stranded DNA-cellulose suggest that the X. laevis DNA helicase is different from previously described eukaryotic DNA helicases.     

Cysteine 111 affects coupling of single-stranded DNA binding to ATP hydrolysis in the herpes simplex virus type-1 origin-binding protein

Herpes simplex virus type-1 origin-binding protein (UL9 protein) initiates viral replication by unwinding the origins. It possesses sequence-specific DNA-binding activity, single-stranded DNA-binding activity, DNA helicase activity, and ATPase activity that is strongly stimulated by single-stranded DNA. We have examined the role of cysteines in its action as a DNA helicase. The DNA helicase and DNA-dependent ATPase activities of UL9 protein were stimulated by reducing agent and specifically inactivated by the sulfhydryl-specific reagent N-ethylmaleimide. To identify the cysteine responsible for this phenomenon, a conserved cysteine in the vicinity of the ATP-binding site (cysteine 111) was mutagenized to alanine. UL9C111A protein exhibits defects in its DNA helicase and DNA-dependent ATPase activities and was unable to support origin-specific DNA replication in vivo. A kinetic analysis indicates that these defects are due to the inability of single-stranded DNA to induce high affinity ATP binding in UL9C111A protein. The DNA-dependent ATPase activity of UL9C111A protein is resistant to N-ethylmaleimide, while its DNA helicase activity remains sensitive. Accordingly, sensitivity of UL9 protein to N-ethylmaleimide is due to at least two cysteines. Cysteine 111 is involved in coupling single-stranded DNA binding to ATP-binding and subsequent hydrolysis, while a second cysteine is involved in coupling ATP hydrolysis to DNA unwinding.     

Inhibition of unwinding and ATPase activities of pea MCM6 DNA helicase by actinomycin and nogalamycin

Pea mini-chromosome maintenance 6 (MCM6) single subunit (93 kDa) forms homohexamer (560 kDa) and contains an ATP-dependent and replication fork stimulated 3′ to 5′ DNA unwinding activity along with intrinsic DNA-dependent ATPase and ATP-binding activities¹ (Plant Mol Biol 2010; DOI: 10.1007/s11103-010-9675-7). Here, we have determined the effect of various DNA-binding agents, such as actinomycin, nogalamycin, daunorubicin, doxorubicin, distamycin, camptothecin, cyclophosphamide, ellipticine, VP-16, novobiocin, netropsin, cisplatin, mitoxantrone and genistein on the DNA unwinding and ATPase activities of the pea MCM6 DNA helicase. The results show that actinomycin and nogalamycin inhibited the DNA helicase (apparent Ki values of 10 and 1 µM, respectively) and ATPase (apparent Ki values of 100 and 17 µM, respectively) activities. Although, daunorubicin and doxorubicin also inhibited the DNA helicase activity of pea MCM6, but with less efficiency; however, these could not inhibit the ATPase activity. These results suggest that the intercalation of the inhibitors into duplex DNA generates a complex that impedes translocation of MCM6, resulting in the inhibitions of the activities. This study could be useful in our better understanding of the mechanism of plant nuclear DNA helicase unwinding.Key words: ATPase, actinomycin, DNA-binding agents, DNA helicase, nogalamycin, pea MCM6     

Flavones inhibit the hexameric replicative helicase RepA

Helicases couple the hydrolysis of nucleoside triphosphates (NTPs) to the unwinding of double-stranded nucleic acids and are essential in DNA metabolism. Thus far, no inhibitors are known for helicases except heliquinomycin isolated from Streptomyces sp. As the three-dimensional structure of the hexameric replicative DNA helicase RepA encoded by the broad host-range plasmid RSF1010 is known, this protein served as a model helicase to search for inhibitory compounds. The commercially available flavone derivatives luteolin, morin, myricetin and dimyricetin (an oxidation product of myricetin) inhibited the ATPase and double-stranded DNA unwinding activities of RepA. Dimyricetin was the most effective inhibitor for both activities. Single-stranded DNA-dependent RepA ATPase activity is inhibited non-competitively by all four compounds. This finding contrasts the inhibition of phosphoinositide 3-kinase by flavones that fit into the ATP binding pocket of this enzyme. Myricetin also inhibited the growth of a Gram-positive and a Gram-negative bacterial species. As we found other hexameric and non-hexameric prokaryotic helicases to be differentially sensitive to myricetin, flavones may provide substructures for the design of molecules helpful for unraveling the mechanism of helicase action and of novel pharmacologically useful molecules.     

Further characterization of DNA helicase activity of mouse DNA-dependent adenosinetriphosphatase B (DNA helicase B)

The DNA helicase activity of DNA-dependent ATPase B purified from mouse FM3A cells [Seki, M., Enomoto, T., Hanaoka, F., & Yamada, M. (1987) Biochemistry 26, 2924-2928] has been further characterized. The helicase activity was assayed with partially duplex DNA substrates in which oligonucleotides to be released by the enzyme were radiolabeled. Oligonucleotides with or without phosphate at the 5' termini or with a deoxy- or dideoxyribose at the 3'-terminal nucleotides were displaced by this enzyme with essentially the same efficiency and with the same ATP (and dATP) and Mg2+ requirements. Thus, there was no strict structure requirement for both ends of duplex regions of substrates to be unwound by the enzyme. Shorter strands were released more readily than longer strands up to the length of 140 bases. The attachment of the enzyme to a single-stranded DNA region was a prerequisite for the neighboring duplex to be unwound; the enzyme-catalyzed unwinding was inhibited competitively by the coaddition of single-stranded DNAs which act as cofactors of the ATPase activity. Their activities as the inhibitor of helicase were well correlated with those as the cofactor of ATPase. The helicase B was found to migrate along single-stranded DNA in the 5' to 3' direction by the use of single strands with short duplex regions at both 3' and 5' ends as substrate. A possible role of this enzyme in DNA replication in mammalian cells is discussed.     

DNA helicase from calf thymus. Purification to apparent homogeneity and biochemical characterization of the enzyme

We have purified a DNA helicase from calf thymus to apparent homogeneity by monitoring the activity with a strand displacement assay. DNA helicase followed the DNA polymerase alpha-primase complex through chromatography on phosphocellulose and hydroxylapatite. Separation from DNA polymerase alpha-primase complex as well as from the bulk of another DNA-dependent ATPase was achieved on heparin-Sepharose. Further purification steps included ATP-agarose and fast protein liquid chromatography-Mono S. A 47-kDa polypeptide cosedimented with the DNA helicase activity in a glycerol gradient as well as in gel filtration on Superose 6. The calf thymus DNA helicase had a sedimentation coefficient of 4-7 S and Stokes radius of about 45 A suggesting that the enzyme might be monomer in its functional form. DNA helicase activity requires a divalent cation with Mg2+ being more efficient than Mn2+ or Ca2+. Hydrolysis of ATP is required since the two nonhydrolyzable ATP analogs adenosine 5'-O-(3-thiotriphosphate) and adenylyl (beta, gamma-methylene)diphosphonate cannot substitute for ATP or dATP in the displacement reaction. Calf thymus DNA helicase is able to use ATP, dATP, dideoxy-ATP, CTP, and dCTP with Km for ATP and dATP of 0.2 and 0.25 mM, respectively. The enzyme can displace a fragment of 24 bases completely in an enzyme concentration- and time-dependent manner. The DNA helicase appears to bind to single-stranded DNA and to move to single-strand double-strand transition. The directionality of unwinding is 3'----5' with respect to the single-stranded DNA to which the enzyme is bound.     

DNA-dependent adenosinetriphosphatase B from mouse FM3A cells has DNA helicase activity

We have detected at least four forms of DNA-dependent ATPase in mouse FM3A cell extracts [Tawaragi, Y., Enomoto, T., Watanabe, Y., Hanaoka, F., & Yamada, M. (1984) Biochemistry 23, 529-533]. The purified fraction of one of the four forms, ATPase B, has been shown to have DNA helicase activity by using a DNA substrate which permits the detection of limited unwinding of the helix. The DNA substrate consists of single-stranded circular fd DNA and the hexadecamer complementary to the fd DNA, which bears an oligo(dT) tail at the 3' terminus. The helicase activity and DNA-dependent ATPase activity cosedimented at 5.5 S on glycerol gradient centrifugation. The helicase required a divalent cation for activity (Mg2+ congruent to Mn2+ greater than Ca2+). The optimal concentrations of these divalent cations were 5 mM. The requirement of divalent cations of the DNA helicase activity was very similar to that for the DNA-dependent ATPase activity of ATPase B. The helicase activity was absolutely dependent on the presence of a nucleoside triphosphate. ATP was the most effective cofactor among the ribo- and deoxyribonucleoside triphosphates tested, and considerable levels of helicase activity were observed with other ribo- and deoxyribonucleoside triphosphates. The efficiency of a nucleoside triphosphate to serve as cofactor for the helicase activity correlated with the capacity of the nucleotide to serve as substrate for the DNA-dependent ATPase activity. The nonhydrolyzable ATP analogues such as adenosine 5'-O-(3-thiotriphosphate) were not effective for the helicase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure-Based Mutagenesis Study of Hepatitis C Virus NS3 Helicase

The NS3 protein of hepatitis C virus (HCV) is a bifunctional protein containing a serine protease in the N-terminal one-third, which is stimulated upon binding of the NS4A cofactor, and an RNA helicase in the C-terminal two-thirds. In this study, a C-terminal hexahistidine-tagged helicase domain of the HCV NS3 protein was expressed in Escherichia coli and purified to homogeneity by conventional chromatography. The purified HCV helicase domain has a basal ATPase activity, a polynucleotide-stimulated ATPase activity, and a nucleic acid unwinding activity and binds efficiently to single-stranded polynucleotide. Detailed characterization of the purified HCV helicase domain with regard to all four activities is presented. Recently, we published an X-ray crystallographic structure of a binary complex of the HCV helicase with a (dU)(8) oligonucleotide, in which several conserved residues of the HCV helicase were shown to be involved in interactions between the HCV helicase and oligonucleotide. Here, site-directed mutagenesis was used to elucidate the roles of these residues in helicase function. Four individual mutations, Thr to Ala at position 269, Thr to Ala at position 411, Trp to Leu at position 501, and Trp to Ala at position 501, produced a severe reduction of RNA binding and completely abolished unwinding activity and stimulation of ATPase activity by poly(U), although the basal ATPase activity (activity in the absence of polynucleotide) of these mutants remained intact. Alanine substitution at Ser-231 or Ser-370 resulted in enzymes that were indistinguishable from wild-type HCV helicase with regard to all four activities. A mutant bearing Phe at Trp-501 showed wild-type levels of basal ATPase, unwinding activity, and single-stranded RNA binding activity. Interestingly, ATPase activity of this mutant became less responsive to stimulation by poly(U) but not to stimulation by other polynucleotides, such as poly(C). Given the conservation of some of these residues in other DNA and RNA helicases, their role in the mechanism of unwinding of double-stranded nucleic acid is discussed.     

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.     

极端嗜热古菌——芝田硫化叶菌DNA解旋酶的分离纯化和性质研究

采用Qsepharose离子交换层析、磷酸纤维素P1 1吸附层析、肝素琼脂糖吸附层析、Su perdex 2 0 0凝胶过滤和PhenylSuperose疏水层析等步骤 ,从嗜酸热芝田硫化叶菌细胞裂解液中分离纯化了一个DNA解旋酶。该解旋酶具有受DNA激活的ATP酶活性。根据SDS PAGE测定结果 ,该酶的分子质量约为 63kD。芝田硫化叶菌DNA解旋酶可以解开底物上 70bp的双链区 ,其解旋活性依赖于双链区旁的单链分叉。该解旋酶的活性依赖于Mg2 + 和ATP的水解 ,在NaCl浓度超过 2 0 0mmol L时受到抑制。该酶的最适pH为 6 7。该酶在 40℃～ 80℃之间均有活性 ,70℃时活性最高。芝田硫化叶菌DNA解旋酶是从古菌中分离得到的第一个天然DNA解旋酶。