A novel DNA helicase from calf thymus.

We report the purification and characterization of a novel DNA helicase from calf thymus tissue. This enzyme partially copurifies with DNA polymerase epsilon* through many of the chromatographic procedures used to isolate it. The enzyme contains an intrinsic DNA-dependent ATPase activity. It can displace short oligonucleotides annealed to long single stranded substrates, in an ATP-dependent reaction. Use of this assay indicates that the DNA helicase translocates in a 3' to 5' direction with respect to the substrate strand to which it is bound. Maximal efficiency of displacement is accomplished by hydrolysis of (d)ATP as cofactor, however, (d)CTP can also be utilized resulting in a 5-fold decrease in the level of displacement. Displacement activity is enhanced by the presence of saturating amounts of Escherichia coli single stranded DNA-binding protein, not affected by the presence of phage T4 gene 32 protein, and inhibited by human replication factor A. The DNA helicase has a molecular mass of approximately 104 kDa as measured by denaturing gel electrophoresis, and an S value of 5.4 obtained from glycerol gradient sedimentation. Direct [alpha-32P]ATP cross-linking labels a protein of molecular mass approximately 105 kDa, providing further evidence that this polypeptide contains the helicase active site. In view of the differences in the properties of this helicase from four others recently identified in calf and designated A through D, we propose the name helicase E.     

The Escherichia coli dnaB replication protein is a DNA helicase

Genetic and biochemical analyses indicate that the Escherichia coli dnaB replication protein functions in the propagation of replication forks in the bacterial chromosome. We have found that the dnaB protein is a DNA helicase that is capable of unwinding extensive stretches of double-stranded DNA. We constructed a partially duplex DNA substrate, containing two preformed forks of single-stranded DNA, which was used to characterize this helicase activity. The dnaB helicase depends on the presence of a hydrolyzable ribonucleoside triphosphate, is maximally stimulated by a combination of E. coli single-stranded DNA-binding protein and E. coli primase, is inhibited by antibody directed against dnaB protein, and is inhibited by prior coating of the single-stranded regions of the helicase substrate with the E. coli single-stranded DNA-binding protein. It was determined that the dnaB protein moves 5' to 3' along single-stranded DNA, apparently in a processive fashion. To invade the duplex portion of the helicase substrate, the dnaB protein requires a 3'-terminal extension of single-stranded DNA in the strand to which it is not bound. Under optimal conditions at 30 degrees C, greater than 1 kilobase pair of duplex DNA can be unwound within 30 s. Based on these findings and other available data, we propose that the dnaB protein is the primary replicative helicase of E. coli and that it actively and processively migrates along the lagging strand template, serving both to unwind the DNA duplex in advance of the leading strand and to potentiate synthesis by the bacterial primase of RNA primers for the nascent (Okazaki) fragments of the lagging strand.     

Characterization of Werner syndrome protein DNA helicase activity: directionality, substrate dependence and stimulation by replication protein A.

Werner syndrome is an inherited disease characterized by premature aging, genetic instability and a high incidence of cancer. The wild type Werner syndrome protein (WRN) has been demonstrated to exhibit DNA helicase activity in vitro. Here we report further biochemical characterization of the WRN helicase. The enzyme unwinds double-stranded DNA, translocating 3'-->5' on the enzyme-bound strand. Hydrolysis of dATP or ATP, and to a lesser extent hydrolysis of dCTP or CTP, supports WRN-catalyzed strand-displacement. K m values for ATP and dATP are 51 and 119 microM, respectively, and 2.1 and 3.9 mM for CTP and dCTP, respectively. Strand-displacement activity of WRN is stimulated by single-stranded DNA-binding proteins (SSBs). Among the SSBs from Escherichia coli, bacteriophage T4 and human, stimulation by human SSB (human replication protein A, hRPA) is the most extensive and occurs with a stoichiometry which suggests direct interaction with WRN. A deficit in the interaction of WRN with hRPA may be associated with deletion mutations that occur at elevated frequency in Werner syndrome cells.     

DNA substrate specificity of DNA helicase E from calf thymus.

DNA helicase E from calf thymus has been characterized with respect to DNA substrate specificity. The helicase was capable of displacing DNA fragments up to 140 nucleotides in length, but was unable to displace a DNA fragment 322 nucleotides in length. DNA competition experiments revealed that helicase E was moderately processive for translocation on single strand M13mp18 DNA, and that the helicase would dissociate and rebind during a 15 minute reaction. Comparison of the rate of ATPase activity catalyzed by helicase E on single strand DNA substrates of different lengths, suggested a processivity consistent with the competition experiments. The helicase displayed a preference for displacing primers whose 5' terminus was fully annealed as opposed to primers with a 12 nucleotide 5' unannealed tail. The presence of a 12 nucleotide 3' tail had no effect on the rate of displacement. DNA helicase E was capable of displacing a primer downstream of either a four nucleotide gap, a one nucleotide gap or a nick in the DNA substrate. Helicase E was inactive on a fully duplex DNA 30 base pairs in length. Calf thymus RP-A stimulated the DNA displacement activity of helicase E. These properties are consistent with a role for DNA helicase E in chromosomal DNA repair.     

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).     

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.     

A Saccharomyces cerevisiae DNA helicase associated with replication factor C.

A novel DNA helicase has been isolated from Saccharomyces cerevisiae. This DNA helicase co-purified with replication factor C (RF-C) during chromatography on S-Sepharose, DEAE-silica gel high performance liquid chromatography (HPLC), Affi-Gel Blue-agarose, heparin-agarose, single-stranded DNA-cellulose, fast protein liquid chromatography MonoS, and hydroxyapatite HPLC. Surprisingly, the helicase could be separated from RF-C by sedimentation on a glycerol gradient in the presence of 200 mM NaCl. The helicase is probably a homodimer of a 60-kDa polypeptide, which by UV cross-linking has been shown to bind ATP. It has a single-stranded DNA-dependent ATPase activity, with a Km for ATP of 60 microM. The DNA helicase activity depends on the hydrolysis of NTP (dNTP), with ATP and dATP the most efficient cofactors, followed by CTP and dCTP. The DNA helicase has a 5' to 3' directionality and is only marginally stimulated by coating the single-stranded DNA with the yeast single-stranded DNA-binding protein RF-A.     

Calf thymus alkaline phosphatase. I. Properties of the membrane-bound enzyme.

A membrane fraction from calf thymocytes was used to investigate molecular and catalytic properties of membrane-bound alkaline phosphatase (ortho-phosphoric-monoester phosphohydrolase EC 3.1.3.1). The principal findings were: 1. Solubilization of membranes with the non-ionic detergent Triton X-100 increases alkaline phosphatase activity by 30-40%. The enzyme activity elutes in a single peak (Stokes' radius = 7.7 nm) after chromatography in Sepharose 6B in the presence of Triton X-100. The activity also sediments as a single component of approx. 6.4 S during centrifugation in sucrose gradients containing Triton X-100. 2. Ion-exchange chromatography and isoelectric focusing in the presence of Triton X-100 indicate substantial charge heterogeneity. Two overlapping bands, a peak at pH 5.92 with a pronounced shoulder at pH 5.29, are apparent by isoelectric focusing. 3. The pH optimum for hydrolysis of p-nitrophenylphosphate (pNPhP) by the undissolved enzyme(s) is 9.57. Half-maximal activity occurs at pH 8.65 and ph 10.45. Triton X-100 has no effect on the pH profile. 4. Catalytic activity is affected by amines, especially analogues of ethanolamine. Diethanolamine exerts a unique stimulatory effect, but does not change the pH dependency. Increasing the concentration of diethanolamine from 0 to 1 M causes a 6-fold increase in Km and a 10-fold increase in the rate of hydrolysis of pNPhP. Glycine is inhibitory. 5. EDTA causes an irreversible loss of activity with t1/2 (1 mM EDTA, pH 8.2, 23 degrees C) = 3.5 h. Optimal activity is achieved in 0.1--1.0 mM Mg2+, although this does not cause the degree of activation reported to occur with the purified enzymes. Other divalent ions are inhibitory. Concentrations required to reduce activity to 50% of control are: Zn2+, 4.0 muM (no added Mg2+) and 30 muM (in the presence of 1 mM Mg2+); Mn2+, 0.25 mM (+/- Mg2+); Ca2+, 20 mM (+/- Mg2+). 6. Monovalent cations have little effect on activity. In the absence of added Mg2+, 50--150 mM Na+ is partially inhibitory, but markedly less so in the presence of 1 mM Mg2+. K+ has no significant effect. 7. Of the substrates tested, pNPhP (Km = 44 muM) was most rapidly hydrolyzed. Other substrates (rate relative to pNPhP) were alpha-naphthylphosphate (0.79), 2'-AMP (0.80), 5'-AMP (0.70), 3'-AMP (0.63), alpha-glycerophosphate (0.47) and glucose 6-phosphate (0.35). Phosphodiesterase activity was less than or equal to 10% of the phosphomonoesterase activity (for pNPhP) as evidenced by the lack of hydrolysis of bis(p-nitrophenyl)-phosphate and cyclic 3',5'-AMP. The ability of these substances to inhibit hydrolysis of pNPhP reflected their capacity as substrates, i.e. the most inhibitory were the most rapidly hydrolyzed.     

Bacteriophage-T7-induced DNA-priming protein. A novel enzyme involved in DNA replication.

The T7gene-4 protein has been purified to near homogeneity using a complementation assay in vitro, and it is designated T7 DNA-priming protein (DNA primase). The purified enzyme enables T7 DNA polymerase to initate DNA synthesis on various circular single-stranded DNA templates by a mechanism which involes the synthesis of a very short RNA primer. The oligoribonucleotide, which is linked to the product DNA via a 3':5'-phosphodiester bond, starts with pppA-C and terminates predominantly with AMP. When only ATP and CPT are precursors, the RNA primer is found to be primarily a tetranucleotide of the sequence pppA-C-C-A. Using oligoribonucleotides in place of ribonucleoside triphosphates as chain initators, T7 DNA-priming protein drastically increases the efficiency with which T7 DNA polymerase can utilize particular tetranucleotide primers containing A and C residues. T7 DNA-priming protein also enables T7 DNA polymerase to make use of native or nicked duplex T7 DNA as template-primer. This reaction does not require ribonucleoside triphosphates, although their addition enhances DNA synthesis 2--4 fold. The product formed in their absence is covalently attached to the template DNA and is found to contain a few long branches when examined by electron microscopy. In the presence of ribonucleoside triphosphates most of the newly made product arises from imitation of DNA chains de novo. Incubation of three proteins: T7 DNA-priming protein, T7 DNA polymerase, and T7 DNA-binding protein, with ribonucleoside and deoxyribonucleoside triphosphates, and with phiX174DNA as template leads to the generation of 'rolling circle-like' structures as visualized in the electron microscope. Single-stranded regions at the tail-circle junction indicate that initations can occur de novo on the displaced complementary strand. This is consistent with a discontinuous mode of 'lagging' strand synthesis and suggests that the same proteins may also be responsible for fork propagation in vivo.     

A geminivirus replication protein is a sequence-specific DNA binding protein.

The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two circular DNA molecules designated as components A and B. The A component encodes the only viral protein, AL1, that is required for viral replication. We showed that AL1 interacts specifically with TGMV A and B DNA by using an immunoprecipitation assay for AL1:DNA complex formation. In this assay, a monoclonal antibody against AL1 precipitated AL1:TGMV DNA complexes, whereas an unrelated antibody failed to precipitate the complexes. Competition assays with homologous and heterologous DNAs established the specificity of AL1:DNA binding. AL1 produced by transgenic tobacco plants and by baculovirus-infected insect cells exhibited similar DNA binding activity. The AL1 binding site maps to 52 bp on the left side of the common region, a 235-bp region that is highly conserved between the two TGMV genome components. The AL1:DNA binding site does not include the putative hairpin structure that is conserved in the common regions or the equivalent 5' intergenic regions of all geminiviruses. These studies demonstrate that a geminivirus replication protein is a sequence-specific DNA binding protein, and the studies have important implications for the role of this protein in virus replication.     

A prepriming DNA replication enzyme of Escherichia coli. I. Purification of protein n': a sequence-specific, DNA-dependent ATPase

Protein n', an enzyme essential for in vitro conversion of single-stranded phiX174 DNA to the duplex replicative form, has been purified about 16,000-fold from Escherichia coli. The enzyme is a single polypeptide chain with a native molecular weight of 76,000; about 70 enzyme molecules are present in an E. coli cell. Nearly homogeneous preparations display an ATPase (dATPase) activity which depends on a unique sequence in the phiX174 DNA. Replicative activity of n' protein and its phiX174 DNA-dependent ATPase activity were present in a constant ratio during the latter stages of purification, upon sedimentation in a glycerol gradient, and during heat inactivation. Further studies of the properties of protein n' are presented in a succeeding paper.     

A DNA helicase purified by replication protein A (RPA) affinity chromatography from mouse FM3A cells.

In an effort to identify cellular helicases that mimic the action of SV40 large T-antigen, we performed replication protein A (RPA) affinity chromatography on cell extracts from the mouse mammary carcinoma cell line FM3A. In this way, a novel DNA helicase was isolated and purified to near homogeneity. The most purified fractions showed the presence of two proteins of 28 and 21 kDa. Both proteins interacted with 32P-labeled partially duplex DNA when bound to nitrocellulose membranes and were efficiently UV crosslinked to [alpha-32P]dATP. Helicase activity was strongly stimulated by RPA on DNA substrates containing duplex regions longer than 18 bp. Only weak stimulation was observed in the presence of Escherichia coli single strand DNA binding protein (SSB). The enzyme unwinds DNA in the 5'-3' direction in relation to the strand to which it binds. Only ATP and dATP were efficient as nucleoside triphosphate co-factors, and showed similar Km values of approximately 0.6 mM. The properties of this enzyme suggest that it may take part in reactions mediated by RPA such as those predicted to occur at replication forks or alternatively may function during DNA repair or recombination.     

A prepriming DNA replication enzyme of Escherichia coli. II. Actions of protein n': a sequence-specific, DNA-dependent ATPase

Protein n' of Escherichia coli is required for formation of the prepriming complex in replication of the single-stranded circle of phiX174 DNA. The protein, purified to near homogeneity, possesses ATPase (dATPase) activity in the presence of single-stranded, but not duplex, DNAs. Except for phiX174 DNA, ATPase activity is completely suppressed by coating the DNA with single strand binding protein (SSB). phiX174 DNA possesses a unique sequence with a potential hairpin structure that is recognized as an effector (Shlomai, J., and Kornberg, A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 799-803). Sequences with secondary structure in SSB-coated M13 DNA which are recognized by RNA polymerase, and in coated G4 DNA by primase, are inert for protein n'. Approximately 30 of the 180 molecules of SSB bound to phiX DNA are destabilized by protein n' in an ATP-dependent reaction. These actions by protein n' may be important in recognizing an origin for forming the prepriming complex that leads to initiation of phiX complementary strand synthesis.     

Plasmid replication initiator protein RepD increases the processivity of PcrA DNA helicase

The replication initiator protein RepD encoded by the Staphylococcus chloramphenicol resistance plasmid pC221 stimulates the helicase activity of the Bacillus stearothermophilus PcrA DNA helicase in vitro. This stimulatory effect seems to be specific for PcrA and differs from the stimulatory effect of the Escherichia coli ribosomal protein L3. Whereas L3 stimulates the PcrA helicase activity by promoting co-operative PcrA binding onto its DNA substrate, RepD stimulates the PcrA helicase activity by increasing the processivity of the enzyme and enables PcrA to displace DNA from a nicked substrate. The implication of these results is that PcrA is the helicase recruited into the replisome by RepD during rolling circle replication of plasmids of the pT181 family.     

DNA helicase requirements for DNA replication during bacteriophage T4 infection.

The lytic bacteriophage T4 uses multiple mechanisms to initiate the replication of its DNA. Initiation occurs predominantly at replication origins at early times of infection, but there is a switch to genetic recombination-dependent initiation at late times of infection. The T4 insertion-substitution system was used to create a deletion in the T4 dda gene, which encodes a 5'-3' DNA helicase that stimulates both DNA replication and recombination reactions in vitro. The deletion caused a delay in T4 DNA synthesis at early times of infection, suggesting that the Dda protein is involved in the initiation of origin-dependent DNA synthesis. However, DNA synthesis eventually reached nearly wild-type levels, and the final number of phages produced per bacterium was similar to that of the wild type. When the dda mutant phage also contained a mutation in T4 gene 59 (a gene normally required only for recombination-dependent DNA replication), essentially no DNA was synthesized. Recent in vitro studies have shown that the gene 59 protein loads a component of the primosome, the T4 gene 41 DNA helicase, onto DNA. A molecular model for replication initiation is presented that is based on our genetic data.     

Human DNA helicase B (HDHB) binds to replication protein A and facilitates cellular recovery from replication stress

Maintenance of genomic stability in proliferating cells depends on a network of proteins that coordinate chromosomal replication with DNA damage responses. Human DNA helicase B (HELB or HDHB) has been implicated in chromosomal replication, but its role in this coordinated network remains undefined. Here we report that cellular exposure to UV irradiation, camptothecin, or hydroxyurea induces accumulation of HDHB on chromatin in a dose- and time-dependent manner, preferentially in S phase cells. Replication stress-induced recruitment of HDHB to chromatin is independent of checkpoint signaling but correlates with the level of replication protein A (RPA) recruited to chromatin. We show using purified proteins that HDHB physically interacts with the N-terminal domain of the RPA 70-kDa subunit (RPA70N). NMR spectroscopy and site-directed mutagenesis reveal that HDHB docks on the same RPA70N surface that recruits S phase checkpoint signaling proteins to chromatin. Consistent with this pattern of recruitment, cells depleted of HDHB display reduced recovery from replication stress.     

Bacteriophage T4 UvsW protein is a helicase involved in recombination, repair and the regulation of DNA replication origins.

Bacteriophage T4 UvsW protein is involved in phage recombination, repair and the regulation of replication origins. Here, we provide evidence that UvsW functions as a helicase. First, expression of UvsW allows growth of an (otherwise inviable) Escherichia coli recG rnhA double mutant, consistent with UvsW being a functional analog of the RecG helicase. Second, UvsW contains helicase sequence motifs, and a substitution (K141R) in the Walker 'A' motif prevents growth of the E.coli recG rnhA double mutant. Third, UvsW, but not UvsW-K141R, inhibits replication from a T4 origin at which persistent RNA-DNA hybrids form and presumably trigger replication initiation. Fourth, mutations that inactivate UvsW and endonuclease VII (which cleaves DNA branches) synergistically block repair of double-strand breaks. These in vivo results are consistent with a model in which UvsW is a DNA helicase that catalyzes branch migration and dissociation of RNA-DNA hybrids. In support of this model, a partially purified GST/UvsW fusion protein, but not a GST/UvsW-K141R fusion, displays ssDNA-dependent ATPase activity and is able to unwind a branched DNA substrate.     

Rep protein as a helicase in an active, isolatable replication fork of duplex phi X174 DNA

Rep protein as a helicase combines its actions with those of gene A protein and single-stranded DNA binding protein to separate the strands of phi X174 duplex DNA and thereby can generate and advance a replication fork (Scott, J. F., Eisenberg, S., Bertsch, L. L., and Kornberg, A. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 193-197). Tritium-labeled rep protein is bound in an active gene A protein. phi X174 closed circular duplex supercoiled DNA complex in a 1:1 ratio. Catalytic separation of the strands of the duplex by rep protein, as measured by incorporation of tritium-labeled single-stranded DNA binding protein, requires ATP at a Km value of 8 microM, and hydrolyzes two molecules of ATP for every base pair melted. When coupled to replication in the synthesis of single-strand viral circles, a "looped" rolling-circle intermediate is formed that can be isolated in an active form containing gene A protein, rep protein, single-stranded DNA binding protein, and DNA polymerase III holoenzyme. Unlike the binding of rep protein to single-stranded DNA, where its ATPase activity is distributive, binding to the replicating fork is not affected by ATP, further suggesting a processive action linked to gene A protein. Limited tryptic hydrolysis of rep protein abolishes its replicative activity without affecting significantly its binding of ATP and its ATPase action on single-stranded DNA. These results augment earlier findings by describing the larger role of rep proteins as a helicase, linked in a complex ith other proteins, at the replication fork of a duplex DNA.     

Purification and characterization of phiX174 gene A protein. A multifunctional enzyme of duplex DNA replication.

Synthesis of phiX174 viral (+) strand circles in vitro requires gene A protein, rep protein, DNA binding protein, and DNA polymerase III holoenzyme (Eisenberg, S., Scott, J. F., and Kornberg, A., (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3151-3155). We have used this reaction as an assay to isolate gene A protein in greater than 90% purity. Its molecular weight under denaturing conditions is 59,000. The protein tends to aggregate and lose activity at low ionic strength. Tritium-labeled gene A protein cleaves the phiX174 duplex replicative form and is bound to it in a 1:1 ratio as part of an active replication complex. The attachment, at the 5' phosphoryl end of the cleavage point, is apparently covalent. The complex was not dissociated by: (i) banding in CsCl, (ii) treatment with 0.2 M NaOH, or (iii) boiling in 1% sodium dodecyl sulfate and electrophoresis on a sodium dodecyl sulfate-acrylamide gel; only micrococcal nuclease digestion of the DNA released the protein.