Identification of human DNA helicase V with the far upstream element-binding protein

The properties of human DNA helicase V (HDH V) were studied in greater detail following an improved purification procedure. From 450 g of cultured cells, <0.1 mg of pure protein was isolated. HDH V unwinds DNA unidirectionally by moving in the 3′ to 5′ direction along the bound strand in an ATP- and Mg²⁺-dependent fashion. The enzyme is not processive and can also unwind partial RNA–RNA duplexes such as HDH IV and HDH VIII. The M_r determined by SDS–PAGE (66 kDa) corresponds to that measured under native conditions, suggesting that HDH V exists as a monomer in the nucleus. Microsequencing of the purified HDH V shows that this enzyme is identical to the far upstream element-binding protein (FBP), a protein that stimulates the activity of the c-myc gene by binding specifically to the ‘FUSE’ DNA region localized upstream of its promoter. The sequence of HDH V/FBP contains RGG motifs like HDH IV/nucleolin, HDH VIII/G3BP as well as other human RNA and DNA helicases identified by other laboratories.     

Human DNA helicase II: a novel DNA unwinding enzyme identified as the Ku autoantigen.

Human DNA helicase II (HDH II) is a novel ATP-dependent DNA unwinding enzyme, purified to apparent homogeneity from HeLa cells, which (i) unwinds exclusively DNA duplexes, (ii) prefers partially unwound substrates and (iii) proceeds in the 3' to 5' direction on the bound strand. HDH II is a heterodimer of 72 and 87 kDa polypeptides. It shows single-stranded DNA-dependent ATPase activity, as well as double-stranded DNA binding capacity. All these activities comigrate in gel filtration and glycerol gradients, giving a sedimentation coefficient of 7.4S and a Stokes radius of approximately 46 A, corresponding to a native molecular weight of 158 kDa. The antibodies raised in rabbit against either polypeptide can remove from the solution all the activities of HDH II. Photoaffinity labelling with [alpha-32P]ATP labelled both polypeptides. Microsequencing of the separate polypeptides of HDH II and cross-reaction with specific antibodies showed that this enzyme is identical to Ku, an autoantigen recognized by the sera of scleroderma and lupus erythematosus patients, which binds specifically to duplex DNA ends and is regulator of a DNA-dependent protein kinase. Recombinant HDH II/Ku protein expressed in and purified from Escherichia coli cells showed DNA binding and helicase activities indistinguishable from those of the isolated protein. The exclusively nuclear location of HDH II/Ku antigen, its highly specific affinity for double-stranded DNA, its abundance and its newly demonstrated ability to unwind exclusively DNA duplexes, point to an additional, if still unclear, role for this molecule in DNA metabolism.     

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.     

Stimulation of the DNA unwinding activity of human DNA helicase II/Ku by phosphorylation

The Ku autoantigen is a heterodimeric protein of 70- and 83-kDa subunits, endowed with duplex DNA end-binding capacity and DNA helicase activity (Human DNA Helicase II, HDH II). HDH II/Ku is well established as the DNA binding component, the regulatory subunit as well as a substrate for the DNA-dependent protein kinase DNA-PK, a complex involved in the repair of DNA double-strand breaks and in V(D)J recombination in eukaryotes. The effects of phosphorylation by this kinase on the helicase activity of Escherichia coli-produced HDH II/Ku were studied. The rate of DNA unwinding by recombinant HDH II/Ku heterodimer is stimulated at least fivefold upon phosphorylation by DNA-PK_cs. This stimulation is due to the effective transfer of phosphate residues to the helicase rather than the mere presence of the complex. In vitro dephosphorylation of HeLa cellular HDH II/Ku caused a significant decrease in the DNA helicase activity of this enzyme.     

Human DNA helicase IV is nucleolin, an RNA helicase modulated by phosphorylation

The cDNA encoding human DNA helicase IV (HDH IV), a 100-kDa protein which unwinds DNA in the 5′ to 3′ direction with respect to the bound strand, was cloned and sequenced. It was found to be identical to the human cDNA encoding nucleolin, a ubiquitous eukaryotic protein essential for pre-ribosome assembly. HDH IV/nucleolin can unwind RNA-RNA duplexes, as well as DNA-DNA and DNA-RNA duplexes. Phosphorylation of HDH IV/nucleolin by cdc2 kinase and casein kinase II enhanced its unwinding activity in an additive way. The Gly-rich C-terminal domain possesses a limited ATP-dependent duplex-unwinding activity which contributes to the helicase activity of HDH IV/nucleolin.     

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).     

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).     

The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3).

To assess the RNA helicase activity of hepatitis C virus (HCV) nonstructural protein 3 (NS3), a polypeptide encompassing amino acids 1175 to 1657, which cover only the putative helicase domain, was expressed in Escherichia coli by a pET expression vector. The protein was purified to near homogeneity and assayed for RNA helicase activity in vitro with double-stranded RNA substrates prepared from a multiple cloning sequence and an HCV 5' nontranslated region (5'-NTR) or 3'-NTR. The enzyme acted successfully on substrates containing both 5' and 3' single-stranded regions (standard) or on substrates containing only the 3' single-stranded regions (3'/3') but failed to act on substrates containing only the 5' single-stranded regions (5'/5') or on substrates lacking the single-stranded regions (blunt). These results thus suggest 3' to 5' directionality for HCV RNA helicase activity. However, a 5'/5' substrate derived from the HCV 5'-NTR was also partially unwound by the enzyme, possibly because of unique properties inherent in the 5' single-stranded regions. Gel mobility shift analyses demonstrated that the HCV NS3 helicase could bind to either 5'- or 3'-tailed substrates but not to substrates lacking a single-stranded region, indicating that the polarity of the RNA strand to which the helicase bound was a more important enzymatic activity determinant. In addition to double-stranded RNA substrates, HCV NS3 helicase activity could displace both RNA and DNA oligonucleotides on a DNA template, suggesting that HCV NS3 too was disposed to DNA helicase activity. This study also demonstrated that RNA helicase activity was dramatically inhibited by the single-stranded polynucleotides. Taken altogether, our results indicate that the HCV NS3 helicase is unique among the RNA helicases characterized so far.     

Characterization of the HeLa cell single-stranded DNA-dependent ATPase/DNA helicase II

A single-stranded DNA-dependent ATPase activity, consisting of two subunits of 83 kDa (p90) and 68 kDa (p70), was previously purified from HeLa cells (Vishwanatha, J.K. and Baril, E.F. (1990) Biochem 29, 8753–8759). Homology of the two subunits of single-stranded DNA-dependent ATPase with the human Ku protein (Caoet al. (1994) Biochem 33, 8548–8557) and identity of the Ku protein as the human DNA helicase II (Tutejaet al. (1994) EMBO J. 13, 4991–5001) have been reported recently. Using antisera raised against the subunits of the HDH II, we confirm that the Hela single-stranded DNA-dependent ATPase is the HDH II. Similar to the activity reported for Ku protein, ssDNA-dependent ATPase binds to double-stranded DNA and the DNA-protein complex detected by gel mobility shift assay consists of both the ATPase subunits. The p90 subunit is predominantly nuclear and is easily dissociated from chromatin. The p70 is distributed in cytosol and nucleus, and a fraction of the nuclear p70 protein is found to be associated with the nuclear matrix. Both the p90 and p70 subunits of the ATPase are present in G₁ and S phase of the cell cycle and are rapidly degraded in the G₂/M phase of the cell cycle.Abbreviations ssDNA single-stranded DNA - dsDNA double-stranded DNA - ATPase adenosine triphosphatase - HDH II human DNA helicase II - PGK 3-phosphoglycerate kinase     

A DNA helicase from Pisum sativum is homologous to translation initiation factor and stimulates topoisomerase I activity

DNA helicases play an essential role in all aspects of nucleic acid metabolism, by providing a duplex-unwinding function. This is the first report of the isolation of a cDNA (1.6 kb) clone encoding functional DNA helicase from a plant (pea, Pisum sativum). The deduced amino-acid sequence has eight conserved helicase motifs of the DEAD-box protein family. It is a unique member of this family, containing DESD and SRT motifs instead of DEAD/H and SAT. The encoded 45.5 kDa protein has been overexpressed in bacteria and purified to homogeneity. The purified protein contains ATP-dependent DNA and RNA helicase, DNA-dependent ATPase, and ATP-binding activities. The protein sequence contains striking homology with eIF-4A, which has not so far been reported as DNA helicase. The antibodies against pea helicase inhibit in vitro translation. The gene is expressed as 1.6 kb mRNA in different organs of pea. The enzyme is localized in the nucleus and cytosol, and unwinds DNA in the 3' to 5' direction. The pea helicase interacts with pea topoisomerase I protein and stimulates its activity. These results suggest that pea DNA helicase could be an important multifunctional protein involved in protein synthesis, maintaining the basic activities of the cell, and in upregulation of topoisomerase I activity. The discovery of such a protein with intrinsic multiple activity should make an important contribution to our better understanding of DNA and RNA transactions in plants.     

A new RNA helicase isolated from HeLa cells that catalytically translocates in the 3' to 5' direction.

We have purified an RNA helicase to near homogeneity from nuclear extracts of HeLa cells. The enzyme migrated as a 130-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and exhibited a sedimentation coefficient of 6.4 on glycerol gradient centrifugation. The enzyme translocated in a 3' to 5' direction and acted catalytically, displacing at least a 4-fold molar excess of duplex RNA compared with the enzyme added. All eight common nucleoside triphosphates supported RNA helicase activity at relatively low concentrations (Km in values in the 15-20 microM level). In the presence of RNA and some single-stranded DNAs, the RNA helicase hydrolyzed all nucleoside triphosphates to nucleoside diphosphates and inorganic phosphate. The enzyme displaced deoxyribooligonucleotides provided they were hydrogen-bonded to RNA possessing 3' single-stranded regions, but it did not displace ribooligonucleotides hydrogen-bonded to DNA containing 3' single-stranded regions. The enzyme, in the absence of ATP, binds to both single-stranded RNA and DNA, but the amount of complex formed with RNA was 20-fold greater than the complex formed with DNA. In both cases, the complex formed in the absence of ATP was rapidly reversed by the addition of ATP and not by adenyl-5'-yl (beta,gamma-methylene)-diphosphate. We propose that the enzyme can bind to both single-stranded RNA and DNA and hydrolyze ATP, but by virtue of its greater stability on RNA, the enzyme can only translocate on RNA possessing 3' single-stranded regions.     

The novel human DNA helicase hFBH1 is an F-box protein

We have identified a novel DNA helicase in humans that belongs to members of the superfamily I helicase and found that it contains a well conserved F-box motif at its N terminus. We have named the enzyme hFBH1 (human F-box DNA helicase 1). Recombinant hFBH1, containing glutathione S-transferase at the N terminus, was expressed in Sf9 cells and purified. In this report, we show that hFBH1 exhibited DNA-dependent ATPase and DNA unwinding activities that displace duplex DNA in the 3' to 5' direction. The hFBH1 enzyme interacted with human SKP1 and formed an SCF (SKP1/Cullin/F-box) complex together with human Cullin and ROC1. In addition, the SCF complex containing hFBH1 as an F-box protein displayed ubiquitin ligase activity. We demonstrate that hFBH1 is the first F-box protein that possesses intrinsic enzyme activity. The potential role of the F-box motif and the helicase activity of the enzyme are discussed with regard to regulation of DNA metabolism.     

Molecular cloning of a cDNA encoding mouse DNA helicase B, which has homology to Escherichia coli RecD protein, and identification of a mutation in the DNA helicase B from tsFT848 temperature-sensitive DNA replication mutant cells

DNA helicase B is a major DNA helicase in mouse FM3A cells. A temperature-sensitive mutant defective in DNA replication, tsFT848, isolated from FM3A cells, has a heat-labile DNA helicase B. In this study, we purified DNA helicase B from mouse FM3A cells and determined partial amino acid sequences of the purified protein. By using a DNA probe synthesized according to one of the partial amino acid sequences, a cDNA was isolated, which encoded a 121.5 kDa protein containing seven conserved motifs for DNA/RNA helicase superfamily members. A database search revealed similarity between DNA helicase B and the α subunit of exodeoxyribonuclease V of a number of prokaryotes including Escherichia coli RecD protein, but no homologous protein was found in yeast. The cDNA encoding DNA helicase B from tsFT848 was sequenced and a mutation was found between DNA/RNA helicase motifs IV and V.     

Isolation of a DNA helicase from HeLa cells requiring the multisubunit human single-stranded DNA-binding protein for activity.

A DNA helicase, dependent on the multisubunit human single-stranded DNA binding protein (HSSB), was isolated from HeLa cells. At low levels of helicase, only the multisubunit SSBs, HSSB and yeast SSB, stimulated DNA helicase activity. At high levels of the helicase Escherichia coli SSB partially substituted for HSSB whereas other SSBs such as T4 gene 32 and adenovirus DNA binding protein did not stimulate the enzyme activity. Maximal activation of helicase activity occurred in the presence of one molecule of HSSB for every 20 nucleotides of single-stranded DNA. The addition of E. coli SSB significantly lowered the amount of HSSB required for strand displacement, suggesting that the HSSB plays at least two roles in the activation of the helicase. One is to bind single-stranded DNA, thereby preventing sequestration of the helicase, the other involves the interaction of the HSSB with the helicase. Monoclonal antibodies that interact with the 70- and 34-kDa subunits of HSSB inhibited its stimulation of the helicase activity. The DNA helicase acted catalytically in displacing duplex DNA and translocated in the 3' to 5' direction. The helicase displaced fragments from both ends of a DNA substrate that contained duplex region at both termini, but the 3' to 5' fragment was displaced 20 times faster than the 5' to 3' fragment. Since this helicase also displaced fully duplex DNA, the release of the 5' to 3' fragment may have occurred by entry of the helicase through the duplex end in a 3' to 5' direction.     

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.     

The human coronavirus 229E superfamily 1 helicase has RNA and DNA duplex-unwinding activities with 5'-to-3' polarity

The human coronavirus 229E replicase gene encodes a protein, p66HEL, that contains a putative zinc finger structure linked to a putative superfamily (SF) 1 helicase. A histidine-tagged form of this protein, HEL, was expressed using baculovirus vectors in insect cells. The purified recombinant protein had in vitro ATPase activity that was strongly stimulated by poly(U), poly(dT), poly(C), and poly(dA), but not by poly(G). The recombinant protein also had both RNA and DNA duplex-unwinding activities with 5'-to-3' polarity. The DNA helicase activity of the enzyme preferentially unwound 5'-oligopyrimidine-tailed, partial-duplex substrates and required a tail length of at least 10 nucleotides for effective unwinding. The combined data suggest that the coronaviral SF1 helicase functionally differs from the previously characterized RNA virus SF2 helicases.