DNA Unwinding Protein Research Articles

Stimulation of rat liver α- and β-type DNA polymerases by an homologous DNA-unwinding protein

Stimulation of DNA polymerases by homologous DNA-unwinding proteins is one of the strongest arguments to assign a role to these proteins in the in vivo DNA synthesis [I]. In the field of prokaryotic replication, Molineux et al. [2] have shown in E. coli that only DNA polymerase II was stimulated in vitro by the homologous DNA-unwinding protein. Moreover, a significant inhibition of both DNA polymerase I and DNA polymerase III was observed. Nevertheless, Weiner et al. [3] have reported that the DNAunwinding protein was also absolutely required in the conversion of the single stranded DNA of phage G4 into replicative form, catalyzed by DNA polymerase III holoenzyme. In both cases, optimum of stimulation occured with an amount of unwinding protein sufficient to cover all the single stranded regions of the template DNA. In the field of eukaryotic replication, DNAunwinding proteins had been described by Herrick and Alberts in calf thymus [4,5] and by Otto et al. in mouse cells [7]. These proteins are able to stimulate specifically the homologous DNA polymerase CK [6,7]. A DNA-unwinding protein was purified to homogeneity from rat liver and its main properties had been studied. In the native state, the protein is a tetramer

Intracellular DNA-protein complexes from bacteriophage T4-infected cells isolated by a rapid two-step procedure. Characterization and identification of the protein components.

A simple technique has been developed for isolating intracellular DNA and its bound proteins from uninfected and phage-infected bacteria. This technique, which utilizes aqueous salt concentrations in the physiological range, is based upon the fact that DNA exists in normal cell lysates in a stiff random coil conformation, and has an unusually large excluded volume to mass ratio. Such stiff coils display a unique combination of low sedimentation coefficient and large Stokes radius, enabling them to be separated rapidly from all other cellular components by successive centrifugal and gel permeation steps. Analysis of this purified intracellular DNA fraction from bacteriophage T4-infected Escherichia coli reveals mainly DNA and protein, with a small amount of RNA also present. Among the major proteins obtained are the DNA-dependent RNA polymerase of the host and the products of T4 genes rIIA, rIIB, and 32 (DNA-"unwinding" protein). Small amounts of the proteins coded by T4 genes 43 (DNA polymerase) and 42 (dCMP hydroxymethylase) have also been identified, in addition to at least 13 other phage-coded proteins of unidentified genes. Much of the phage-coded protein in the complex, including the gene 32 protein, does not exchange readily with the same protein exogenously added in the lysate.

Interaction of DNA with DNA binding proteins II. Displacement of Escherichia coli DNA unwinding protein and the condensed structure of DNA complexed with protein HD

Three DNA binding proteins from Escherichia coli cells have been complexed with single-stranded phage fd DNA. Electron microscopy reveals granular substructures in the complexes formed with protein HD. In complexes of DNA unwinding protein with fd DNA both protein HD and phage-coded gene 5 protein partially displace the unwinding protein which results in the formation of structures characteristic for the DNA complexes formed with either protein HD or gene 5 protein alone. Combination of protein HD with double-stranded phage T7 DNA leads to a progressive folding and condensing of the genome. The structures observed are discussed in relation to current concepts of the packing of DNA in protein complexes.

Membrane-associated assembly of M13 phage in extracts of virus-infected Escherichia coli.

Assembly of coliphage M13 is known to occur as the viral DNA crosses the cytoplasmic membrane, shedding its virus-coded DNA unwinding protein and acquiring from the membrane approximately 2400 copies of the major coat protein. Conditions are described in which extracts of M13-infected E. coli and membranes prepared from such extracts will support virus assembly at a rate equivalent to that of intact cells. Extracts prepared from cells infected with temperature-sensitive M13 mutants in genes 1, 3, 4, or 5 are temperature-sensitive in this cell-free assembly reaction. Phage assembly in vitro requires magnesium and as yet an unidentified heat-stable cofactor of low molecular weight. The rate of virus assembly is approximately linear with respect to extract concentration over a 10(4)-fold range, consistent with the observation that the entire M13 assembly activity copurifies with the cell membrane fraction.

Interaction of DNA with DNA binding proteins III. Infectivity of protein-complexed phage fd DNA in Escherichia coli spheroplasts

Complex formation of circular, single-stranded phage fd DNA with Escherichia coli DNA binding protein HD or phage fd gene 5 protein keeps infection of E. coli spheroplasts at the level of free phage DNA, whereas complexes of this DNA with E. coli DNA unwinding protein show a strongly reduced efficiency of transfection. Displacement of the unwinding protein by HD protein or gene 5 protein also maintains the poor adsorption of the complexes to spheroplasts. Free E. coli DNA unwinding protein and residual amounts of this protein bound to the DNA may interfere with the adsorption and the uptake of the phage genome.

A mechanism of duplex DNA replication revealed by enzymatic studies of phage phi X174: catalytic strand separation in advance of replication.

The enzyme system for duplicating the duplex, circular DNA of phage phi X174 (replicative form) in stage II of the replicative life cycle was shown to proceed in two steps: synthesis of the viral (+) strand ]stage II(+)], followed by synthesis of the complementary (-) strand ]stage II(-)] [Eisenberg et al. (1976) Proc. Natl. Acad. Sci. USA 73, 3151-3155]. Novel features of the mechanism of the stage II(+) reaction have now been observed. The product, synthesized in extensive net quantities, is a covalently closed, circular, single-stranded DNA. The supercoiled replicative form I template and three of the four required proteins--the phage-induced cistron A protein (cis A), the host rep protein (rep), and the DNA polymerase III holoenzyme (holoenzyme)--act catalytically; the Escherichia coli DNA unwinding (or binding) protein binds the product stoichiometrically. In a reaction uncoupled from replication, cis A, rep, DNA binding protein, ATP, and Mg2+ separate the supercoiled replicative form I into its component single strands coated with DNA binding protein. In the presence of Mg2+, cis A, nicks the replicative form I; rep, ATP, and Mg2+ achieve strand separation with a concurrent cleavage of ATP and binding of DNA binding protein to the single strands. rep exhibits a single-stranded DNA-dependent ATPase activity. These observations suggest that the rep enzymatically melts the duplex at the replicating fork, using energy provided by ATP; this mechanism may apply to the replication of the E. coli chromosome as well.

Studies on the noncooperative binding of the Escherichia coli DNA unwinding protein to single-stranded nucleic acids.

The noncooperative binding of the Escherichia coli DNA unwinding protein to single-stranded DNA oligomers has been studied by means of equilibrium dialysis. Dialyses were performed under a number of solution and temperature conditions using oligomers of varying length and base compositions. The results of these studies, which include a Scatchard analysis of the binding, have allowed us to propose a model for the cooperative binding of the protein to single-stranded DNA. The results of experiments dealing with the interaction of the protein with single-stranded RNA are also presented.

Further studies on the DNA-unwinding protein of the rat ventral prostate: Evidence for local areas of denaturation

Soluble extracts of the rat ventral prostate contain a protein capable of modifying the secondary structure of native (helical) DNA. Two assays for this activity have been developed; the selective retention of [ 3H]-labelled prostate DNA on nitrocellulose membranes and the enhancement of DNA synthesis by the replicative form of prostate DNA polymerase using native DNA as template. Although not purified to homogeneity, the changes in the secondary structures of DNA promoted by this protein cannot be attributed simply to contamination of the preparations with deoxyribonucleases or other enzymes. Because the structural changes evoked in DNA are similar in nature but not extent to those achieved by thermal denaturation, we have termed this prostate constituent a DNA-unwinding protein. In keeping with this concept, three independent methods have been used to demonstrate a limited strand separation in native prostate after treatment with this protein; digestion with S1 nuclease, binding of mithramycin and immunological precipitation by antibodies raised against single-stranded (denatured) DNA.

Crystallization of a DNA-unwinding protein: Preliminary X-ray analysis of fd bacteriophage gene 5 product

The gene 5 protein from bacteriophage fd, which binds to single-stranded progeny fd DNA, was obtained as large single crystals and subjected to X-ray diffraction analysis. The crystals are of monoclinic space group C2 with a = 75.8 A ̊ , b = 28.0 A ̊ , c = 42.5 A ̊ and β = 103 °12′ . The unit cell has one molecule of 9800 daltons as the asymmetric unit.

Enzymatic replication of viral and complementary strands of duplex DNA of phage phiX174 proceeds by seprate mechanisms.

Multiplication of the duplex, circular, phage phiX174DNA (replicative form, RF) in stage II of the replicative life cycle has been observed with a crude enzyme preparation [Eisenberg et al. (1976) Proc, Natl. Acad, Sci. USA 73, 1594-1597]. This stage has now been partially reconstituted with purified proteins and subdivided into two stages: II(+) and II(-). In stage II(+), viral (+) strand synthesis is carried out by four proteins: the phage-induced, cistron A-dependent protein, rep-dependent protein, DNA unwinding protein, and DNA polymerase III holenzyme. In stage II(-), complementary (-) strand synthesis utilizes the product of stage II(+) as template and the multiprotein system previously identified in the stage I synthesis of a complementary strand on the viral DNA template to produce RF. The multiprotein system includes DNA unwinding protein, proteins i and n, dnaB protein, dnaC protein, dnaG protein, and DNA polymerase III holoenzyme. A discussion of these two separate mechanism for synthesis of (+) and (-) strands suggests that they may account for essentially all the replicative stages in the life cycle of phiX174.

Control of mutation frequency by bacteriophage T4 DNA polymerase. I. The CB120 antimutator DNA polymerase is defective in strand displacement.

The ts CB1200 (antimutator) mutation in bacteriophage T4 DNA polymerase increases the accuracy of DNA replication since it results in a decrease in the frequency of mutations in other phage genes. The CB120 polymerases differs from the wild type enzyme in the slow rate at which it copies templates where primer extension requries displacement of polynucleotides base-paired to the template strand, even in the presence of the T4 DNA unwinding protein (gene 32-protein). The ratio of nucleotides turned over (DNA-dependent conversion of deoxynucleoside triphosphate to deoxynucleoside monophosphate) to nucleotides stably incorporated into product is 10 to 100 times higher with the mutant than wild type enzyme, depending on the DNA used as the template. This high turnover rate may increase the efficiency of removal of noncomplementary nucleotides by the antimutator enzyme and is in agreement with the findings of Muzyczka et al, (Muzyczka, N., Poland, R. L., and Bessman, M. J. (1972) J. Biol, Cehm. 247, 7116-7122) with the L141 and L42 antimutator T4 DNA polymerases. Since the 3'- to 5'-exonuclease activity of the CB120 mutant polymerase is not higher than that of the wild type enzyme, it is suggested that the high turnover rate may result from increased opportunity to remove newly incorporated nucleotides due to the slow rate at which the mutant enzyme moves to the next template nucleotide. In the accompanying paper we show that the CB120 antimutator polymerase also initially selects incorrect nucleotides for incorporation less frequently than the wild type enzyme. Thus this antimutator polymerase appears to have both greater accuracy in nucleotide selection and an enhanced ability to remove incorrect nucleotides.

Mutator mutations in bacteriophage T4 gene 32 (DNA unwinding protein).

Bacteriophage T4 gene 32 encodes a DNA unwinding protein required for DNA replication, repair, and recombination. Gene 32 temperature-sensitive mutations enhance virtually all base pair substitution mutation rates.

Enzymic unwinding of DNA. 1. Purification and characterization of a DNA-dependent ATPase from Escherichia coli.

Evidence from various sources in the literature suggests that, in connection with DNA, ATP dephosphorylation can be used to provide energy for mechanical effects. Starting from this concept we have studied a novel DNA-dependent ATPase purified to 90% homogeneity from Escherichia coli. The enzyme has a peptide weight near 180 000 and, in high salt, is a monomeric, probably highly anisometric molecule. In salt-free buffer, where the ATPase activity is highest, the enzyme forms aggregates. ATP is the preferred substrate (Km 0.27 mM) and dephosphorylated at the gamma-position at a maximal rate near 10(4) molecules per enzyme monomer per min at 35 degrees C. A requirement for divalent cation is best satisfied by Mg2+ or Ca2+ and the requirement for DNA best by the single-stranded, circular DNA of phages phiX174 (Km 62 nM nucleotide) and fd indicating that the enzyme recognizes internal DNA regions. When saturated with E. coli DNA unwinding protein phiX DNA is not accepted but, once in contact with the DNA, the enzyme is little inhibited by unwinding protein. Apparently the unwinding protein interferes preferentially with the recognition of DNA. The enzyme does not detectably cleave DNA, and for this and genetic reasons is not identical with the recBC ATPase or the K12 restriction ATPase of the extracted cells. The enzyme is probably not identical either with the dnaB-product-associated ATPase or the ATPase activity found in DNA polymerase III holoenzyme under appropriate conditions, and it is certainly not identical with a DNA-dependent ATPase of molecular weight 69 000 from E. coli which has recently been purified. Attempts to ascribe the enzyme to other genes, including recA, lex and rep, have failed.

Purification and physical characterization of nucleic acid helix-unwinding proteins from calf thymus.

We have devised a general protein fractionation procedure which selects for eukaryotic DNA-binding proteins, some of which resemble DNA-unwinding proteins from prokaryotes. Proteins were selected which (a) pass through a native DNA-cellulose column, (b) bind to a denatured DNA-cellulose column, and (c) remain bound to the latter column during a rinse with a dilute solution of the sodium salt of the polyanion dextran sulfate. When this fractionation was applied to the soluble proteins fo calf thymus, three major protein species were recovered. The predominant one has an apparent molecular weight of about 24,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is isoelectric near neutrality, and elutes as a monomer from denatured DNA-cellulose at moderate NaCl concentrations. This protein, designated calf-unwinding protein 1 (UP1), has been purified to homogeneity. However, isoelectric focusing reveals four or five subspecies (apparently separated by single-charge differences) which differ appreciably in their affinities for DNA. Two other major proteins are obtained which have apparent molecular weights in sodium dodecyl sulfate of 33,000: the first, which elutes with low salt from DNA-cellulose as a homogeneous preparation, appears to be a basic protein (although it is clearly not a histone); the other, which elutes from DNA-cellulose as the major component of a "high salt eluting fraction," is an acidic protein which co-purifies with less prominent species of higher molecular weights. Proteins similar to each of these three major calf thymus proteins have been observed by us and others in tissue culture cells of mouse, hamster, monkey, and humans, suggesting their wide occurrence among eukaryotes.

Studies on the structure of intracellular bacteriophage T4 DNA

We have studied DNA metabolism in cells infected with bacteriophage T4 mutants producing different temperature-sensitive proteins required for T4 DNA replication. In general, the structure of the normal, 600 to 1000 S intracellular DNA is maintained when DNA synthesis is blocked by shifting such mutant-infected cells to a non-permissive temperature. However, in cells infected with a phage producing a thermolabile gene 32 protein (T4 DNA-unwinding protein), we find that the intracellular DNA is completely converted to simple DNA pieces ranging from one-fourth to two genomes in length (39 S to 80 S) within two minutes after a shift to high temperature. This conversion appears to require the presence of an active T4 gene 49 product. We present evidence that these DNA pieces are generated from the normal intracellular DNA by cutting at single-stranded regions uncovered by thermal inactivation of gene 32 protein, and that these single-stranded regions are greatly reduced or missing in a gene 49-deficient infection. The entire conversion can be closely mimicked in vitro by incubation of the intracellular DNA in detergent-treated lysates with the single-strand specific endonuclease, S 1. Striking features are the apparent first-order kinetics with which DNA is removed by S 1 nuclease from the normal rapidly sedimenting conformation, and the absence of appreciable quantities of normally sedimenting, oligomeric DNA intermediates in this breakdown process. The data suggests a “brush-like” model for the intracellular T4 chromosome, with the DNA held at roughly genome-length intervals by some non-DNA core material resistant to both strong detergent and protease treatments.

Single-stranded DNA structure and DNA polymerase activity in the presence of nucleic acid helix-unwinding proteins from calf thymus.

In the preceding articles we have described the isolation and some of the properties of two calf thymus proteins which bind selectively to single-stranded DNA and which appear analogous to previously isolated prokaryotic DNA-unwinding proteins. In the present work we demonstrate two further points of analogy. First, both the calf UP1 and the high salt eluting proteins form protein-rich complexes with single-stranded DNA, and hold this DNA in a rigid, extended conformation. Second, these proteins stimulate the calf thymus DNA polymerase-alpha; phage T4 gene 32-protein does not. The stimulation of a homologous DNA polymerase is characteristic of several prokaryotic DNA-unwinding proteins and is assumed to reflect their in vivo role in DNA synthesis.

Isolation of an intermediate which precedes dnaG RNA polymerase participation in enzymatic replication of bacteriophage phi X174 DNA.

Conversion of phi X174 single-stranded DNA to the duplex replicative form (RF) in vitro requires at least 10 purified proteins. Three stages - strand initiation, elongation, and termination - comprise this conversion. We now identify a separate stage in strand initiation which precedes dnaG RNA polymerase participation. Incubation of five proteins - protein i, protein n, DNA unwinding protein, dnaB protein, and dnaC protein - with ATP and phi X174 DNA forms an intermediate which enables subsequent stages measured by DNA synthesis to proceed 20 times faster. The intermediate can be isolated in quantitative yield by gel filtration or by ultracentrifugation. Protein i and protein n are required in less than stoichiometric amounts and appear to be absent from the isolated intermediate. Whereas formation of the intermediate is sensitive to antibody to protein i and to N-ethylmaleimide (an inhibitor of protein n and dnaC protein), the intermediate itself is resistant to these reagents. DNA unwinding protein complexes the DNA in a ratio of 60 molecules per circle. Synthesis of the intermediate appears to require stoichiometric quantities of dnaB protein and dnaC PROTEin but their presence in the intermediate has not been established as yet.

A DNA fragment from the origin of single-strand to double-strand DNA replication of bacteriophage fd.

A specific complex is formed between fd DNA, Escherichia coli DNA unwinding protein, and RNA polymerase (EC 2.7.7.6; nucleosidetriphosphate:RNA nucleotidyltransferase) during the first steps in the conversion of the single-stranded viral DNA to the double-stranded replicative form. In this complex a unique DNA fragment of about 120 nucleotides is protected against nuclease digestion. Both the requirements for its isolation and its position on the map of the phage genome indicate that the fragment contains the origin of single-strand to double-strand DNA replication. The isolated DNA fragment possesses double-strand-like characteristics, which protect it from being covered by the DNA unwinding protein and thus indirectly positions the RNA polymerase to the origin of replication.

Studies on the cooperative binding of the Escherichia coli DNA unwinding protein to single-stranded DNA.

The cooperative binding of the Escherichia coli DNA unwinding protein to single-stranded DNA has been studied by electron microscopy. Analysis of the electron microscopic data by means of a simple statistical mechanical model has yielded a value of 3.8-7.6 X 10(10) l./mol for the cooperative binding constant in 0.15 M NaCl. Studied under elevated salt conditions have shown that the average DNA protein complex length is 50% of the length found at 0.04 or 0.15 M NaCl.

De novo synthesis of a polymer of deoxyadenylate and deoxythymidylate by calf thymus DNA polymerase alpha.

In a reaction mixture containing calf thymus DNA polymerase alpha (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase; EC 2.7.7.7), calf thymus DNA unwinding protein, DNA, deoxyadenosine 5'-triphosphate and deoxythymidine 5'-triphosphate, a copolymer of deoxyadenylate and deoxythymidylate is synthesized after a lag period of 1-2 hr. In the presence of the four deoxyribonucleoside triphosphates only deoxyadenylate and deoxythymidylate are incorporated into the polymer and the rate of synthesis is decreased. The reaction variably occurs in the absence of DNA or DNA unwinding protein but with a greatly entended lag period. The optimal Mg2+ concentration for synthesis of the polymer of deoxyadenylate and deoxythymidylate is 1 mM, in contrast to an optimal Mg2+ concentration of 8 mM for DNA synthesis with activated DNA as template. Characterization of the product of de novo synthesis indicates that it is the alternating copolymer, poly(dA-dT).