A DNA polymerase from Ustilago maydis. 1. Purification and properties of the polymerase activity.

A DNA polymerase from Ustilago maydis has been purified to apparent homogeneity. The native enzyme possesses a subunit structure consisting of 50000 and 55000-dalton monomers. The apparent sedimentation coefficient of the polymerase activity in the absence of salt is 8.4 S (Mr=180000-200000), that in its presence (0.6 M NaCl or 0.12 M KCl) being 6.3 S (Mr=80000-100000). Low concentrations of EDTA also converted the 8.4-S to a 6.3-S form, whereas magnesium ions catalysed the reverse association. The enzyme has an absolute requirement for both a DNA or RNA template and a DNA primer. For homopolymer templates the primer requirement was satisified by a short complementary oligodeoxynucleotide, but oligoribonucleotides were extremely inefficient primers. With the template-primer poly(dA) X (dT)12, the enzyme added an average of 50 dTMP nucleotides on to each primer molecule, whereas with poly(rA) X (dT)12, this figure was 300. The enzyme also possesses an associated deoxyribonuclease activity. No other DNA polymerase activity was detected in cell-free extracts of U. maydis.     

Characterization of the 5'' to 3'' exonuclease associated with Thermus aquaticus DNA polymerase.

Thermus aquaticus DNA polymerase was shown to contain an associated 5' to 3' exonuclease activity. Both polymerase and exonuclease activities cosedimented with a molecular weight of 72,000 during sucrose gradient centrifugation. Using a novel in situ activity gel procedure to simultaneously detect these two activities, we observed both DNA polymerase and exonuclease in a single band following either nondenaturing or denaturing polyacrylamide gel electrophoresis: therefore, DNA polymerase and exonuclease activities reside in the same polypeptide. As determined by SDS-polyacrylamide gel electrophoresis this enzyme has an apparent molecular weight of 92,000. The exonuclease requires a divalent cation (MgCl2 or MnCl2), has a pH optimum of 9.0 and excises primarily deoxyribonucleoside 5'-monophosphate from double-stranded DNA. Neither heat denatured DNA nor the free oligonucleotide (24-mer) were efficient substrates for exonuclease activity. The rate of hydrolysis of a 5'-phosphorylated oligonucleotide (24-mer) annealed to M13mp2 DNA was about twofold faster than the same substrate containing a 5'-hydroxylated residue. Hydrolysis of a 5'-terminal residue from a nick was preferred threefold over the same 5'-end of duplex DNA. The 5' to 3' exonuclease activity appeared to function coordinately with the DNA polymerase to facilitate a nick translational DNA synthesis reaction.     

Deoxyribonucleic acid polymerases of BHK-21/C13 cells. Partial purification and characterization of the enzymes.

DNA polymerase from BHK-21/C13 cells were separated into two species, DNA polymerase I corresponding to the heterogeneous enzyme with sedimentation coefficient of 6-8S, and DNA polymerase II, corresponding to the enzyme with sedimentation coefficient of 3.3S. DNA polymerase I was purified 114-fold and DNA polymerase II 154-fold by a simple extraction procedure followed by column chromatography on phosphocellulose and gel filtration through Sephadex G-100. The purified enzymes differed markedly in respect of pH optimum, stimulation and inhibition by K+, Km for the deoxyribonucleoside 5'-triphosphates, stability to heating at 45 degrees C, and inhibition by N-ethylmaleimide. The preferred primer-template for both enzymes was "activated" DNA (DNA submitted to limited degradation by pancreatic deoxyribonuclease); native or thermally denatured DNA templates were relatively very poorly copied. When certain synthetic templates were tested, substantial differences were revealed between the two enzymes. Poly[d(A-T)] was poorly used by polymerase I but was superior to "activated" DNA for polymerase II. Poly[d(A)]-oligo[d(pT)10] was used efficiently by polymerase I but not by polymerase II. Poly(A)-oligo[d(pT)10] was not an effective primer-template although polymerase I could use it to a limited extent when Mn2+ replaced Mg2+ in the polymerase reaction and when the temperature of incubation was lowered from 37 degrees to 30 degrees C. When only one or two or three triphosphates were supplied in the reaction mixture, the activity of polymerase I was more severly diminished than that of polymerase II.     

T7-induced DNA polymerase. Characterization of associated exonuclease activities and resolution into biologically active subunits.

Bacteriophage T7-induced DNA polymerase has been isolated by a procedure suitable for large scale use and which yields near homogeneous enzyme. In addition to previously described DNA polymerase activity and 3' to 5' exonucleolytic activity on single stranded DNA (Grippo, P., and Richardson, C. C. (1971) J. Biol. Chem. 246, 6867-6873), the enzyme also possesses a highly active exonuclease which hydrolyzes duplex substrates with 3' to 5' directionality. The native polymerase has been dissociated using 6 M guanidine HCl and resolved into biologically active subunits: T7 gene 5 protein and Escherichia coli thioredoxin. The phage-specified subunit obtained by this procedure is deficient in DNA polymerase and double strand exonuclease activities, with deficiencies in these activities being apparent at the level of a single turnover. However, it possesses near normal levels of a single strand hydrolytic activity which is identical to that associated with the native polymerase with respect to substrate specificity and suppression of hydrolysis by low levels of deoxyribonucleoside 5'-triphosphates. Thioredoxin forms a molecular complex with the T7 gene 5 protein, and addition of the host protein restores restores DNA polymerase and double strand exonuclease activities to near normal levels.     

The activity of deoxyribonucleic acid polymerase in some species of algae

1. The activities of DNA polymerase preparations from the algae Euglena gracilis, Chlamydomonas reinhardtii, Chlorella pyrenoidosa, Anabaena variabilis and Anacystis nidulans were measured. The blue-green algae Anabaena and Anacystis contain a 5-20-fold higher activity of the enzyme than do the green algae. DNA polymerases from the blue-green algae show a pH optimum of 9 and prefer a relatively low Mg(2+) concentration (1-3mm). DNA polymerases from the green algae, however, display a pH optimum between 7.5 and 8.5 and an optimum Mg(2+) concentration of 8mm. With all algae, a higher polymerase activity was obtained with denatured salmon sperm DNA as template than with native DNA. All four deoxyribonucleoside 5'-triphosphates must be present for full activity of the polymerases. 2. With one exception, the deoxyribonuclease activities in the preparations, measured under conditions of the DNA polymerase assay, are low compared with corresponding preparations from Escherichia coli. Chlamydomonas extracts contain a high deoxyribonuclease activity. 3. After purification on columns of DEAE-cellulose, the polymerase activity was linear over a wide range of protein concentrations, except for Chlamydomonas preparations, where the observed deviation from linearity was probably attributable to the high nuclease activity. 4. DNA polymerases from all these algae bind strongly to DNA-cellulose; 6-40-fold purifications of the enzyme were obtained by chromatography on columns of DNA-cellulose. 5. The partially purified polymerases of Euglena and Anacystis are heat-labile but become much more heat-stable when tested in the presence of DNA.     

DNA polymerases from Chlamydomonas reinhardii. Further characterization, action of inhibitors and associated nuclease activities.

The properties of three DNA polymerase species A, B and C, purified from Chlamydomonas reinhardii were compared. DNA polymerases A and B have Km values with respect to deoxyribonucleoside triphosphates of 19 micron and 3 micron respectively. DNA polymerase A is most active with activated DNA, but will also use native DNA and synthetic RNA and DNA templates with DNA primers. DNA polymerase B is also most active with activated DNA, but will use denatured DNA and synthetic DNA templates. It is inactive with RNA templates. DNA polymerase B is completely inactive in the presence of 100 micron-heparin, which has no effect on DNA polymerase A activity. Heparin dissociates DNA polymerase B into subunits that are still catalytically active, but which heparin inhibited. DNA polymerase B possesses deoxyribonuclease activity that is inhibited by 5 micron-heparin, suggesting that the deoxyribonuclease is an integral part of the DNA polymerase moiety. DNA polymerase A is devoid of nuclease activity. DNA polymerase C is similar to DNA polymerase B in all these properties, though it is more active with RNA primers and has greater heat-sensitivity.     

Characterization of an RNA-directed DNA polymerase found in association with murine intracytoplasmic A-particles.

An RNA-directed DNA polymerase was found to be associated with intracytoplasmic A-particles from DBA/2 mouse leukemia cells. The enzyme activity was detected after disrupting the purified particles with 2 M NaCl-20 mM dithiothreitol. The presence of a divalent cation and all four deoxyribonucleoside triphosphates was essential for this enzyme activity. The enzyme had a clear preference for Mg2+ over Mn2+. Cesium sulfate isopycnic gradient centrifugation of the DNA product synthesized in the actinomycin D-containing reaction revealed the presence of DNA-RNA hybrid. Furthermore, the purified DNA product was found to hybridize with RNA isolated from A-particles. These observations strongly indicate that the endogenous A-particle RNA serves as the template for the DNA polymerase.     

Identification and characterization of a 3' to 5' exonuclease associated with spinach chloroplast DNA polymerase.

Spinach chloroplast DNA polymerase was shown to copurify with a 3' to 5' exonuclease activity during DEAE-cellulose, hydroxylapatite, and heparin-agarose column chromatography. In addition, both activities comigrated during nondenaturing polyacrylamide gel electrophoresis and cosedimented through a glycerol gradient with an apparent molecular weight of 105,000. However, two forms of exonuclease activity were detected following velocity sedimentation analysis. Form I constituted approximately 35% of the exonuclease activity and was associated with the DNA polymerase, whereas the remaining activity (form II) was free of DNA polymerase and exhibited a molecular weight of approximately 26,500. Resedimentation of form I exonuclease generated both DNA polymerase associated and DNA polymerase unassociated forms of the exonuclease, suggesting that polymerase/exonuclease dissociation occurred. The exonuclease activity (form I) was somewhat resistant to inhibition by N-ethylmaleimide, whereas the DNA polymerase activity was extremely sensitive. Using in situ detection following SDS-polyacrylamide activity gel electrophoresis, both form I and II exonucleases were shown to reside in a similar, if not identical, polypeptide of approximately 20,000 molecular weight. Both form I and II exonucleases were equally inhibited by NaCl and required 7.5 mM MgCl2 for optimal activity. The 3' to 5' exonuclease excised deoxyribonucleoside 5'-monophosphates from both 3'-terminally matched and 3'-terminally mismatched primer termini. In general, the exonuclease preferred to hydrolyze mismatched 3'-terminal nucleotides as determined from the Vmax/Km ratios for all 16 possible combinations of matched and mismatched terminal base pairs. These results suggest that the 3' to 5' exonuclease may be involved in proofreading errors made by chloroplast DNA polymerase.     

Characterization of the Bacillus subtilis bacteriophage PBS2-induced DNA polymerase and its associated exonuclease activity.

The DNA polymerase induced by Bacillus subtilis bacteriophage PBS2 has a Stokes radius of 7.2 in buffers of high ioninc strength, suggesting a molecular weight in the range 145,000 to 195,000. The polypeptide bands observed on gel electrophoresis in dodecyl sulfate have apparent molecular weights of 78,000 and 69,000 (and possibly another 27,000) in equimolar amounts. In buffers of low ionic strength, the enzyme appears to form large aggregates and even precipitates, with about 90% loss of activity. A nuclease activity co-purifies with the PBS2 DNA polymerase and shows similar responses to changes in pH, MgCl2, N-ethylmaleimide, temperature, and dextran sulfate levels. The nuclease produces deoxyribonucleoside 5'monophosphates from denatured DNA containing thymine or uracil. No endonuclease activity is detectable on supercoiled DNA. The inhibition of nuclease activity by added deoxyribonucleoside triphosphates, the DNA-dependent turnover of triphosphates, to free monophosphates during DNA polymerization, the inhibition of nuclease activity by 3'-phosphates on the DNA template-primer, and the pattern of digestion of 5'-[32P]phosphate-labeled DNA all indicate that the PBS2 DNA polymerase-associated hydrolytic activity is a 3' leads to 5'-exonuclease.     

Deoxyribonucleic acid nucleotidyltransferase from Landschütz ascites-tumour cells. Partial purification and general properties

1. A purification procedure for DNA nucleotidyltransferase from Landschütz ascites-tumour cells is described. The enzyme can be separated from endogenous nucleic acid and from triphosphatase and deoxyribonuclease activities measurable at pH7.5. 2. The basic properties of the nucleotidyltransferase reaction are as follows. The enzyme has optimum activity at pH7.2-7.4. It displays an absolute requirement for DNA-primer, thermally-denatured DNA serving three to ten times as efficiently in this respect as native DNA. Maximum synthesis of polydeoxyribonucleotide occurs in the presence of all four deoxyribonucleoside 5'-triphosphates, but a limited incorporation of mononucleotide into polynucleotide is observed when the system is provided with only one triphosphate, or with various combinations of mono-, di- and tri-phosphates. The reaction requires the presence of a bivalent cation, and of those tested, Mg(2+) ions were by far the most effective. Manganous ions promoted synthesis but to a much smaller extent. Calcium ions did not support synthesis at all. At the appropriate concentrations, the univalent cations (sodium and potassium) stimulated the reaction by 25% and 125% respectively. The presence of EDTA in the reaction mixture stimulates the system five- to ten-fold. 3. The storage characteristics of the enzyme (as well as the activities of the various fractions) improve markedly if EDTA and 2-mercaptoethanol are included in the enzyme solution and in all preparative buffer solutions. 4. The enzyme loses more than 95% of its activity after heating for 1min. at 45 degrees . If the heating is conducted in the presence of DNA, the enzyme becomes relatively heat-resistant (presumably as a consequence of complex-formation with the DNA) and may actually display an activation effect. This is discussed in relation to a possible molecular conformation of the enzyme. 5. The product of the nucleotidyltransferase reaction is precipitable by acid or ethanol, and is susceptible to the actions of deoxyribonucleases I and II, snake-venom and spleen phosphodiesterases, and micrococcal nuclease. It forms a band in a density gradient of caesium chloride at a density similar to that of the DNA-primer. 6. By the criteria of nearest-neighbour frequency analyses, the product of the nucleotidyltransferase reaction has the characteristics to be expected of a polynucleotide synthesized in accordance with the template directions of the primer.     

Properties of the 3' to 5' exonuclease associated with porcine liver DNA polymerase gamma. Substrate specificity, product analysis, inhibition, and kinetics of terminal excision.

Porcine liver DNA polymerase gamma was shown previously to copurify with an associated 3' to 5' exonuclease activity (Kunkel, T. A., and Mosbaugh, D. W. (1989) Biochemistry 28, 988-995). The 3' to 5' exonuclease has now been characterized, and like the DNA polymerase activity, it has an absolute requirement for a divalent metal cation (Mg2+ or Mn2+), a relatively high NaCl and KCl optimum (150-200 mM), and an alkaline pH optimum between 7 and 10. The exonuclease has a 7.5-fold preference for single-stranded over double-stranded DNA, but it cannot excise 3'-terminal dideoxy-NMP residues from either substrate. Excision of 3'-terminally mismatched nucleotides was preferred approximately 5-fold over matched 3' termini, and the hydrolysis product from both was a deoxyribonucleoside 5'-monophosphate. The kinetics of 3'-terminal excision were measured at a single site on M13mp2 DNA for each of the 16 possible matched and mismatched primer.template combinations. As defined by the substrate specificity constant (Vmax/Km), each of the 12 mismatched substrates was preferred over the four matched substrates (A.T, T.A, C.G, G.C). Furthermore, the exonuclease could efficiently excise internally mismatched nucleotides up to 4 residues from the 3' end. DNA polymerase gamma was not found to possess detectable DNA primase, endonuclease, 5' to 3' exonuclease, RNase, or RNase H activities. The DNA polymerase and exonuclease activities exhibited dissimilar rates of heat inactivation and sensitivity to N-ethylmaleimide. After nondenaturing activity gel electrophoresis, the DNA polymerase and 3' to 5' exonuclease activities were partially resolved and detected in situ as separate species. A similar analysis on a denaturing activity gel identified catalytic polypeptides with molecular weights of 127,000, 60,000, and 32,000 which possessed only DNA polymerase gamma activity. Collectively, these results suggest that the polymerase and exonuclease activities reside in separate polypeptides, which could be derived from separate gene products or from proteolysis of a single gene product.     

Streptococcus pneumoniae DNA polymerase I lacks 3''-to-5'' exonuclease activity: localization of the 5''-to-3'' exonucleolytic domain.

The Streptococcus pneumoniae polA gene was altered at various positions by deletions and insertions. The polypeptides encoded by these mutant polA genes were identified in S. pneumoniae. Three of them were enzymatically active. One was a fused protein containing the first 11 amino acid residues of gene 10 from coliphage T7 and the carboxyl-terminal two-thirds of pneumococcal DNA polymerase I; it possessed only polymerase activity. The other two enzymatically active proteins, which contained 620 and 351 amino acid residues from the amino terminus, respectively, lacked polymerase activity and showed only exonuclease activity. These two polymerase-deficient proteins and the wild-type protein were hyperproduced in Escherichia coli and purified. In contrast to the DNA polymerase I of Escherichia coli but similar to the corresponding enzyme of Thermus aquaticus, the pneumococcal enzyme appeared to lack 3'-to-5' exonuclease activity. The 5'-to-3' exonuclease domain was located in the amino-terminal region of the wild-type pneumococcal protein. This exonuclease activity excised deoxyribonucleoside 5'-monophosphate from both double- and single-stranded DNAs. It degraded oligonucleotide substrates to a decameric final product.     

Enhanced deoxyribonucleic Acid polymerase activity of chromatin from soybean hypocotyls treated with 2,4-dichlorophenoxyacetic Acid

Chromatin isolated from soybean (Glycine max L., var. Wayne) hypocotyls was capable of catalyzing the polymerization of labeled deoxyribonucleoside triphosphate in the presence of the three other deoxyribonucleoside triphosphates into a trichloroacetic acid-insoluble product. This product was insensitive to base hydrolysis and ribonuclease, but was sensitive to acid hydrolysis and deoxyribonuclease. Chromatin-DNA polymerase required Mg²⁺ and all four deoxyribonucleoside triphosphates for maximal activity. Inorganic pyrophosphate and actinomycin D inhibited the polymerase activity, but 2, 4-dichlorophenoxyacetic acid had no effect in vitro. Chromatin from plants previously treated with 2, 4-dichlorophenoxyacetic acid supported a greater level of DNA synthesis than did chromatin from untreated plants.     

High molecular weight deoxyribonucleic acid polymerase of LF hepatoma. Purification and properties.

A high molecular weight DNA polymerase has been purified from the cytosol of a fast growing hepatoma: LF hepatoma. This enzyme sediments at 11.3 S under polymerization reaction conditions (6 mM KCl) and at 8.3 S in higher salt concentrations (200 mM KCl). In either case, no activity is seen in the 3 to 4 S region where low molecular weight DNA polymerase is found. The purified enzyme has a neutral pH optimum and requires a divalent cation, all four deoxyribonucleoside triphosphates and an initiated DNA template for maximal activity. The synthetic template specificity of LF DNA polymerase has been studied. Although this enzyme cannot copy a polyribonucleotide template, the ribostrand of a synthetic hybrid can be used with low efficiency as an initiator for the synthesis of the complementary deoxyribonucleotide strand. The activity of the purified enzyme is strongly inhibited by thiol-blocking agents. The general properties of LF DNA polymerase are similar to those of high molecular weight mammalian DNA polymerases. In our experimental conditions, the error frequency of this tumoral DNA polymerase was no greater than that made by the purified high molecular weight DNA polymerase of regenerating rat liver.     

Further characterization of a DNA polymerase activity in mouse sperm nuclei.

The presence of a nuclear DNA polymerase in mouse sperm from adult testes has been confirmed and the properties of this enzyme further investigated. This activity was shown to be greatly enhanced by treating the spermatozoa with methanol or ethanol before incubation in the reaction medium or by their addition in small amounts to this medium. It was protected against degradation by nuclear proteases by adding soybean trypsin inhibitor and was stimulated by ATP. It was found to be Mg2+ dependent (optimum concentration: 7.5 mM), DNA dependent, and all four deoxynucleoside triphosphates were needed for optimal reaction. The radioactive acid-precipitable product of polymerization was not eliminated by organic solvents, nor by pronase, ribonuclease or by nuclease S1; however, it was converted to a large extent to acid-soluble products by pancreatic deoxyribonuclease. Since it was only partially solubilized by Triton X-100, it therefore did not appear to be preferentially associated with the nuclear membranes. The activity recovered after incubation depended also on the pH (optimum at pH 8.3) and did not work well in a medium for DNA polymerase alpha. The temperature for maximum incorporation of nucleotides was found to be 32 degrees C and, under our conditions, the reaction was linear for 30 min. The DNA polymerase activity was inhibited by low and high concentrations of KCl. It was not lowered by N-ethylmaleimide or p-hydroxymercuribenzoate; urea slightly stimulated the reaction and this stimulation was reversed by subsequent treatment with N-ethylmaleimide. Actinomycin D (40 mug/ml), ethidium bromide (25--50 muM), netropsin (5--50 mug/ml), and spermidine (0.5--2.5 mM) lowered the polymerization of DNA precursors. The nuclear enzyme could shift from the endogenous template to activated exogenous calf thymus DNA, the resulting nuclear radioactivity being reduced. The endogenous DNP template ability was not increased by deoxyribonuclease activation according to the method of Aposhian and Kornberg (J. Biol. Chem. (1962) 237, 519--525) suggesting that the amount of DNA polymerase associated with chromatin was probably limiting the reaction. The DNA polymerase activity detected in mouse sperm nuclei has numerous properties of low molecular weight DNA polymerases (DNA polymerase beta) reported in several eukaryotic organisms.     

Enzymological characterization of DNA polymerase alpha. Basic catalytic properties processivity, and gap utilization of the homogeneous enzyme from human KB cells.

This report describes the results of our initial enzymological characterization of a homogeneous preparation of DNA polymerase alpha that we have purified from cultured human KB cells. Although the enzyme is most reactive with duplex DNA substrates that contain short gaps (optimally activated) in incubations that require Mg2+, the polymerase possesses the intrinsic capacity to copy the initiated ribohomopolymer template, (A)-n, (dT)-200, at low rates in the presence of Mn2+. Because of the preponderance of DNA polymerase alpha in actively multiplying vertebrate cells, it is probable that this low level of activity comprises the majority of the ribopolymer copying activity that can be detected in crude tissue extracts. The presence of contaminating or associated deoxyribonuclease activities can be excluded from the purified enzyme to levels of 10(-4) to 10(-7) of the polymerase activity. The mechanism of polymerization on activated DNA under optimum conditions is moderately processive, with 11 +/- 5 nucleotides incorporated per polymerization cycle. The polymerase is unable to work at nicks or at short gaps of approximately 20 to 30 nucleotides in length, and it measures a surprisingly invariant effective template length on optimally activated DNA and on DNA molecules that have been gapped to varying extents with Escherichia coli exonuclease III. In the "Appendix" we present an amplification of the theoretical formulation of Bambara et al. (Bambara, R. A., Uyemura, D., and Choi, T. (1978) J. Biol. Chem. 253, 413--423) that permits the use of DNA polymerases with significant associated 3' leads to 5'-exonuclease activities for the accurate measurement of average template lengths (gap sizes) and titration of usable 3'-hydroxyl primer termini in gapped, duplex DNA substrates.     

Deoxyribonucleic acid polymerase of bacteriophage T7. Characterization of the exonuclease activities of the gene 5 protein and the reconstituted polymerase.

Homogeneous gene 5 protein of bacteriophage T7, a subunit of T7 DNA polymerase, catalyzes the stepwise hydrolysis of single-stranded DNA in a 3' leads to 5' direction to yield nucleoside 5'-monophosphates. The gene 5 protein itself does not hydrolyze duplex DNA. However, in the presence of Escherichia coli thioredoxin, the host-specified subunit of T7 DNA polymerase, duplex DNA is hydrolyzed in a 3' leads to 5' direction to yield nucleoside 5'-monophosphates. The apparent Km for thioredoxin in the reaction is 4.8 x 10(-8) M, a value similar to that for the apparent Km of thioredoxin in the complementation assay with gene 5 protein to restore T7 DNA polymerase activity. Both exonuclease activities require Mg2+ and a sulfhydryl reagent for optimal activity, and both activities are sensitive to salt concentration. Deoxyribonucleoside 5'-triphosphates inhibit hydrolysis by both exonuclease activities; hydrolysis of single-stranded DNA by the gene 5 protein is inhibited even in the absence of thioredoxin where there is less than 2% active T7 DNA polymerase. E. coli DNA binding protein (helix destabilizing protein) stimulates the hydrolysis of duplex DNA up to 9-fold under conditions where the hydrolysis of the single-stranded DNA is inhibited 4-fold.     

Structure and synthesis of a lipid-containing bacteriophage. A polynucleotide-dependent polynucleotide-pyrophosphorylase activity in bacteriophage PM2.

A polymerase activity is associated with protein IV, a protein which is associated with the DNA in bacteriophage PM2. The native enzyme unit is probably a dimer. Manganese ions are required for the polymerisation reaction and there is a well-defined Mn2+ optimum at 2.5 mM. The pH optimum is at 8.1, the temperature optimum at 28 degrees C. The activity is a polynucleotide-pyrophosphorylating reaction in the presence of ribo- or deoxyribonucleoside triphosphates. The polymerisation reaction is stimulated in the presence of nuclei- acids or polynucleotides as effectors. The product is not covalently linked to the effector.     

Characterization of an Endogenous RNA-Dependent DNA Polymerase Associated with Murine Intracisternal A Particles

An RNA-dependent DNA polymerase associated with intracisternal A particles has been characterized. The enzyme required Mg(2+) or Mn(2+), dithiothreitol and the presence of all four deoxyribonucleoside triphosphates for the expression of maximal activity. Sensitivity of the endogenous RNA-dependent DNA polymerase activity to a low concentration of pancreatic ribonuclease in the presence of a high concentration of NaCl suggested that the enzyme might be utilizing the A particle endogenous RNA as template. Evidence in support of this was provided by analyses of early and late DNA products of the endogenous reaction by Cs(2)SO(4) isopycnic gradient centrifugation and hybridization of purified 60 to 70S and 35S RNAs of A particles with the purified DNA product.