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.     

A DNA helicase from human cells.

We have initiated the characterization of the DNA helicases from HeLa cells, and we have observed at least 4 molecular species as judged by their different fractionation properties. One of these only, DNA helicase I, has been purified to homogeneity and characterized. Helicase activity was measured by assaying the unwinding of a radioactively labelled oligodeoxynucleotide (17 mer) annealed to M13 DNA. The apparent molecular weight of helicase I on SDS polyacrylamide gel electrophoresis is 65 kDa. Helicase I reaction requires a divalent cation for activity (Mg2+ greater than Mn2+ greater than Ca2+) and is dependent on hydrolysis of ATP or dATP. CTP, GTP, UTP, dCTP, dGTP, dTTP, ADP, AMP and non-hydrolyzable ATP analogues such as ATP gamma S are unable to sustain helicase activity. The helicase activity has an optimal pH range between pH8.0 to pH9.0, is stimulated by KCl or NaCl up to 200mM, is inhibited by potassium phosphate (100mM) and by EDTA (5mM), and is abolished by trypsin. The unwinding is also inhibited competitively by the coaddition of single stranded DNA. The purified fraction was free of DNA topoisomerase, DNA ligase and nuclease activities. The direction of unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound. The enzyme also catalyses the ATP-dependent unwinding of a DNA:RNA hybrid consisting of a radioactively labelled single stranded oligodeoxynucleotide (18 mer) annealed on a longer RNA strand. The enzyme does not require a single stranded DNA tail on the displaced strand at the border of duplex regions; i.e. a replication fork-like structure is not required to perform DNA unwinding. The purification of the other helicases is in progress.     

DNA helicase III from HeLa cells: an enzyme that acts preferentially on partially unwound DNA duplexes.

Human DNA helicase III, a novel DNA unwinding enzyme, has been purified to apparent homogeneity from nuclear extracts of HeLa cells and characterized. The activity was measured by using a strand displacement assay with a 32P labeled oligonucleotide annealed to M13 ssDNA. From 305 grams of cultured cells 0.26 mg of pure protein was isolated which was free of DNA topoisomerase, ligase, nicking and nuclease activities. The apparent molecular weight is 46 kDa on SDS polyacrylamide gel electrophoresis. The enzyme shows also DNA dependent ATPase activity and moves unidirectionally along the bound strand in 3' to 5' direction. It prefers ATP to dATP as a cofactor and requires a divalent cation (Mg2+ > Mn2+). Helicase III cannot unwind either blunt-ended duplex DNA or DNA-RNA hybrids and requires more than 84 bases of ssDNA in order to exert its unwinding activity. This enzyme is unique among human helicases as it requires a fork-like structure on the substrate for maximum activity, contrary to the previously described human DNA helicases I and IV, (Tuteja et al. Nucleic Acids Res. 18, 6785-6792, 1990; Tuteja et al. Nucleic Acids Res. 19, 3613-3618, 1991).     

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。     

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.     

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.     

Purification and properties of human DNA helicase VI.

A novel ATP-dependent DNA unwinding enzyme, called human DNA helicase VI (HDH VI), was purified to apparent homogeneity from HeLa cells and characterized. From 327 g of cultured cells, 0.44 mg of pure enzyme was recovered, free of DNA polymerase, ligase, topoisomerase, nicking and nuclease activities. The enzyme behaves as a monomer having an M(r) of 128 kDa, whether determined with SDS-PAGE, or in native conditions. Photoaffinity labelling with [alpha-32P]ATP labelled the 128 kDa protein. Only ATP or dATP hydrolysis supports the unwinding activity for which a divalent cation (Mg2+ > Mn2+) is required. HDH VI unwinds exclusively DNA duplexes with an annealed portion < 32 bp and prefers a replication fork-like structure of the substrate. It cannot unwind blunt-end duplexes and is inactive also on DNA-RNA or RNA-RNA hybrids. HDH VI unwinds DNA unidirectionally by moving in the 3' to 5' direction along the bound strand.     

DNA helicase IV from HeLa cells.

Human DNA helicase IV, a novel enzyme, was purified to homogeneity from HeLa cells and characterized. The activity was measured by assaying the unwinding of 32P labeled 17-mer annealed to M13 ss DNA. From 440g of HeLa cells we obtained 0.31 mg of pure protein. Helicase IV was free of DNA topoisomerases, DNA ligase and nuclease activities. The apparent molecular weight is 100 kDa. It requires a divalent cation for activity (Mg2+ = Mn2+ = Zn2+) and the hydrolysis of only ATP or dATP. The activity is destroyed by trypsin and is inhibited by 200 mM KCl or NaCl, 100 mM potassium phosphate, 45 mM ammonium sulfate, 5 mM EDTA, 20 microM ss M13 DNA or 20 microM poly [G] (as phosphate). The enzyme unwinds DNA by moving in the 5' to 3' direction along the bound strand, a polarity opposite to that of the previously described human DNA helicase I (Tuteja et al Nucleic Acids Res. 18, 6785-6792, 1990). It requires more than 84 bases of single-stranded DNA in order to exert its unwinding activity and does not require a replication fork-like structure. Like human DNA helicase I the enzyme can also unwind RNA-DNA hybrid.     

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     

The isolation and characterization of an RNA helicase from nuclear extracts of HeLa cells.

An RNA helicase, isolated from nuclear extracts of HeLa cells, displaced duplex RNA in the presence of any one of the eight common nucleoside triphosphates. The unwinding reaction was supported most efficiently by ATP and GTP and poorly by dCTP and dTTP. The enzyme activity, purified 300-fold, contained two major protein bands of 80 and 55 kDa when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All fractions that contained RNA helicase activity also possessed single-stranded RNA-dependent nucleoside triphosphatase activity. Purified RNA helicase fractions displaced a hybrid of U4/U6 RNAs with the same efficiency as it displaced other duplex RNA structures. In contrast, the RNA helicase did not displace duplex RNA/DNA and DNA/DNA structures. Evidence is presented that suggests that this RNA helicase can displace duplex RNA by translocating in both the 3' to 5' and the 5' to 3' directions. The properties of the RNA helicase described here differ from the deaminase RNA unwinding activity described in Xenopus oocytes (Bass, B.L., and Weintraub, H. (1987) Cell 48, 607-613) and from the p68 HeLa RNA helicase (Hirling, H., Scheffner, M., Restle, T., and Stahl, H. (1989) Nature 339, 562-564).     

Human DNA helicase V, a novel DNA unwinding enzyme from HeLa cells.

Using a strand-displacement assay with 32P labeled oligonucleotide annealed to M13 ssDNA we have purified to apparent homogeneity and characterized a novel DNA unwinding enzyme from HeLa cell nuclei, human DNA helicase V (HDH V). This is present in extremely low abundance in the cells and has the highest turnover rate among other human helicases. From 300 grams of cultured cells only 0.012 mg of pure protein was isolated which was free of DNA topoisomerase, ligase, nicking and nuclease activities. The enzyme also shows ATPase activity dependent on single-stranded DNA and has an apparent molecular weight of 92 kDa by SDS-polyacrylamide gel electrophoresis. Only ATP or dATP hydrolysis supports the unwinding activity. The helicase requires a divalent cation (Mg2+ > Mn2+) at an optimum concentration of 1.0 mM for activity; it unwinds DNA duplexes less than 25 bp long and having a ssDNA stretch as short as 49 nucleotides. A replication fork-like structure is not required to perform DNA unwinding. HDH V cannot unwind either blunt-ended duplex DNA or DNA-RNA hybrids; it unwinds DNA unidirectionally by moving in the 3' to 5' direction along the bound strand, a polarity similar to the previously described human DNA helicases I and III (Tuteja et al. Nucleic Acids Res. 18, 6785-6792, 1990; Tuteja et al. Nucleic Acid Res. 20, 5329-5337, 1992) and opposite to that of human DNA helicase IV (Tuteja et al. Nucleic Acid Res. 19, 3613-3618, 1991).     

Escherichia coli helicase II (uvrD) protein can completely unwind fully duplex linear and nicked circular DNA

We have examined the duplex DNA unwinding (helicase) properties of the Escherichia coli helicase II protein (uvrD gene product) over a wide range of protein concentrations and solution conditions using a variety of duplex DNA substrates including fully duplex blunt ended and nicked circular molecules. We find that helicase II protein is able to initiate on and completely unwind fully duplex DNA molecules without the requirement for a covalently attached 3' single-stranded DNA tail. This DNA unwinding activity is dependent upon Mg2+ and ATP and requires that the amount of protein be in excess of that needed to saturate the resulting single-stranded DNA. Unwinding experiments on fully duplex blunt ended DNA with lengths of 341, 849, 1625, and 2671 base pairs indicate that unwinding occurs at the same high ratios of helicase II protein/nucleotide, independent of DNA length (50% unwinding requires approximately 0.6 helicase II monomers/nucleotide in 2.5 mM MgCl2, 10% glycerol, pH 7.5, 37 degrees C). Helicase II protein is also able to unwind completely a nicked circular DNA molecule containing 2671 base pairs. At lower but still high molar ratios of helicase II protein to DNA, duplex DNA molecules containing a single-stranded (ss) region attached to a 3' end of the duplex are preferentially unwound in agreement with the results obtained by S. W. Matson [1986) J. Biol. Chem. 261, 10169-10175). This preferential unwinding of duplex DNA with an attached 3' ssDNA most likely reflects the availability of a high affinity site (ssDNA) with the proper orientation for initiation; however, this may not reflect the type of DNA molecule upon which helicase II protein initiates DNA unwinding in vivo. The effects of changes in NaCl, NaCH3COO, and MgCl2 concentration on the ability of helicase II protein to unwind fully duplex DNA and duplex DNA with a 3' ssDNA tail have also been examined. Although the unwinding of fully duplex and nicked circular DNA molecules reported here occurs at higher helicase II protein to DNA ratios than have been previously used in most studies of this protein in vitro, this activity is likely to be relevant to the function of this protein in vivo since very high levels of helicase II protein accumulate in E. coli during the SOS response to DNA damage (approximately 2-5 x 10(4) copies/cell).(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification and purification of a protein that stimulates the helicase activity of the Escherichia coli Rep protein

A polypeptide (Mr = 15,000) has been purified from Escherichia coli cell extracts that significantly stimulates the duplex DNA unwinding reaction catalyzed by E. coli Rep protein. The Rep helicase unwinding reaction was stimulated by as much as 20-fold, upon addition of the stimulatory protein, using either a 71-base pair or a 343-base pair partial duplex DNA molecule as a substrate. The purified Rep helicase stimulatory protein (RHSP) had no intrinsic helicase activity or ATP hydrolysis activity and did not stimulate the single-stranded DNA-dependent ATP hydrolysis reaction catalyzed by Rep protein. It is likely that RHSP stimulates the Rep helicase unwinding reaction by stoichiometric binding to single-stranded DNA. However, a specific interaction between Rep protein and RHSP cannot be ruled out, since RHSP did not stimulate the duplex DNA unwinding reactions catalyzed by E. coli helicase I or the recently discovered 75-kDa helicase. RHSP did stimulate the duplex DNA unwinding reaction catalyzed by E. coli helicase II. The identification and subsequent purification of RHSP from cell extracts demonstrates the feasibility of using direct helicase assays to purify stimulatory proteins.     

Purification and characterization of West Nile virus nucleoside triphosphatase (NTPase)/helicase: evidence for dissociation of the NTPase and helicase activities of the enzyme

The nucleoside triphosphatase (NTPase)/helicase associated with nonstructural protein 3 of West Nile (WN) virus was purified from cell culture medium harvested from virus-infected Vero cells. The purification procedure included sequential chromatography on Superdex-200 and Reactive Red 120 columns, followed by a concentration step on an Ultrogel hydroxyapatite column. The nature of the purified protein was confirmed by immunoblot analysis using a WN virus-positive antiserum, determination of its NH(2) terminus by microsequencing, and a binding assay with 5'-[(14)C]fluorosulfonylbenzoyladenosine. Under optimized reaction conditions the enzyme catalyzed the hydrolysis of ATP and the unwinding of the DNA duplex with k(cat) values of 133 and 5.5 x 10(-3) s(-1), respectively. Characterization of the NTPase activity of the WN virus enzyme revealed that optimum conditions with respect to the Mg(2+) requirement and the monovalent salt or polynucleotide response differed from those of other flavivirus NTPases. Initial kinetic studies demonstrated that the inhibition (or activation) of ATPase activity by ribavirin-5'-triphosphate is not directly related to changes in the helicase activity of the enzyme. Further analysis using guanine and O(6)-benzoylguanine derivatives revealed that the ATPase activity of WN virus NTPase/helicase may be modulated, i.e., increased or reduced, with no effect on the helicase activity of the enzyme. On the other hand the helicase activity could be modulated without changing the ATPase activity. Our observations show that the number of ATP hydrolysis events per unwinding cycle is not a constant value.     

Escherichia coli DNA helicase I catalyzes a unidirectional and highly processive unwinding reaction

Helicase I has been purified to greater than 95% homogeneity from an F+ strain of Escherichia coli, and characterized as a single-stranded DNA-dependent ATPase and a helicase. The duplex DNA unwinding reaction requires a region of ssDNA for enzyme binding and concomitant nucleoside 5'-triphosphate hydrolysis. All eight predominant nucleoside 5'-triphosphates can satisfy this requirement. Unwinding is unidirectional in the 5' to 3' direction. The length of duplex DNA unwound is independent of protein concentration suggesting that the unwinding reaction is highly processive. Kinetic analysis of the unwinding reaction indicates that the enzyme turns over very slowly from one DNA substrate molecule to another. The ATP hydrolysis reaction is continuous when circular partial duplex DNA substrates are used as DNA effectors. When linear partial duplex substrates are used ATP hydrolysis is barely detectable, although the kinetics of the unwinding reaction on linear partial duplex substrates are identical to those observed using a circular partial duplex DNA substrate. This suggests that ATP hydrolysis fuels continuous translocation of helicase I on circular single-stranded DNA while on linear single stranded DNA the enzyme translocates to the end of the DNA molecule where it must slowly dissociate from the substrate molecule and/or slowly associate with a new substrate molecule, thus resulting in a very low rate of ATP hydrolysis.     

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.     

Branch migration of Holliday junctions: identification of RecG protein as a junction specific DNA helicase.

The product of the recG gene of Escherichia coli is needed for normal recombination and DNA repair in E. coli and has been shown to help process Holliday junction intermediates to mature products by catalysing branch migration. The 76 kDa RecG protein contains sequence motifs conserved in the DExH family of helicases, suggesting that it promotes branch migration by unwinding DNA. We show that RecG does not unwind blunt ended duplex DNA or forked duplexes with short unpaired single-strand ends. It also fails to unwind a partial duplex (52 bp) classical helicase substrate containing a short oligonucleotide annealed to circular single-stranded DNA. However, unwinding activity is detected when the duplex region is reduced to 26 bp or less, although this requires high levels of protein. The unwinding proceeds with a clear 3' to 5' polarity with respect to the single strand bound by RecG. Substantially higher levels of unwinding are observed with substrates containing a three-way duplex branch. This is attributed to RecG's particular affinity for junction DNA which we demonstrate would be heightened by single-stranded DNA binding protein in vivo. Reaction requirements for unwinding are the same as for branch migration of Holliday junctions, with a strict dependence on hydrolysis of ATP. These results define RecG as a new class of helicase that has evolved to catalyse the branch migration of Holliday junctions.