Serological analysis of human deoxyribonucleic acid polymerases. Preparation and properties of antiserum to deoxyribonucleic acid polymerase I from human lymphoid cells.

The preparation and properties of an antiserum to human DNA polymerase I (6 to 8 S) are described. Care was taken in the purification of the antigen to remove certain other DNA polymerases found in human cells. An incubation of antigen and antiserum lasting about 48 hours is necessary to achieve maximal inhibition. About 1 mug of the antipolymerase immunoglobulin G, prepared in rats, neutralizes 60% of the activity present in 54 ng of the enzyme. Tritrations varying both antiserum and enzyme demonstrate clear regions of antigen and antibody excess. Inhibition of enzyme activity is about the same whether the templateprimer is (dA)n-(dT)12-18, or partially digested DNA. An assay was developed which measures the remaining activity in the supernatant after precipitation of enzyme-antibody complexes with goat anti-rat immunoglobulin G. In this assay, 2.2 mug of the antipolymerase immunoglobulin G quantitatively bind 33 ng of DNA polymerase I. With use of the direct neutralization assay and the immuno-precipitation test, we found little, if any, antigenic relationship between DNA polymerase I and DNA polymerase II (3.4 S). Similarly, little, if any, relationship was found to the DNA polymerases from five RNA tumor viruses. The activities of RNA-directed DNA polymerases from the blood leukocytes of two patients with acute myelogenous leukemia and from the placentas of rhesus monkeys were not inhibited in neutralization assays which were shortened because these enzymes were thermolabile. In identically shortened neutralization assays, the antipolymerase immunoglobulin G neutralized up to 76% of the activity of DNA polymerase I. In addition to its utility in distinguishing cellular DNA polymerases, the rat antiserum should be useful reagent for testing of novel DNA polymerases isolated in small quantities from human tumors for contamination with DNA polymerase I. This enzyme is present in abundance in proliferating tissue and often confuses the biochemical characterization of these novel enzymes.     

Purification and properties of deoxyribonucleic acid polymerase from Bacillus stearothermophilus.

Deoxyribonucleic acid polymerase I was purified from Bacillus stearothermophilus to 50 to 70% homogeneity. Its molecular weight was 76,000. The enzyme was insensitive to sulfhydryl blocking agents and showed maximal activity at 60 degrees C, pH 8 to 9, 0.25 M KCl, and 0.02 M MgSO4. The rate of heat inactivation of the deoxyribonucleic acid polymerase followed first-order kinetics with a half-life of 90 min at 60 degrees C; the addition of 0.05% bovine serum albumin protected the enzyme, which could be heated for 180 min without loss of activity. The ratios of polymerase to nuclease activities were about 20 for 5'-3' exonuclease and more than 500 for 3'-5' exonuclease. The Km for deoxyribonucleoside-5'-triphosphates was 7 microM.     

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.     

Nuclear and cytoplasmic deoxyribonucleic acid polymerases from rat nephroma cells.

We have examined the DNA polymerases found in a rat nephroma cell line. Using DEAE- and DNA-cellulose chromatography, we have found two major cytoplasmic DNA polymerases and one major and three minor DNA polymerases from the nucleus. The enzymes were all purified, characterized, and distinguished from each other by several criteria. The enzyme require, for maximal activity, a natural or synthetic double-stranded DNA, four deoxynucleoside, triphosphates, and magnesium. They are inhibited to varying degrees by sodium pyrophosphate, ethidium bromide, and rho-chloromercuribenzoate.     

A deoxyribonucleic acid binding protein from KB cells which inhibits deoxyribonuclease activity on single-stranded DNA.

A deoxyribonuclease inhibitor has been purified from KB cells by chromatography on single-stranded DNA-cellulose. Polyacrylamide gel electrophoresis showed the purified preparation to contain two major polypeptides in sodium dodecyl sulfate, with molecular weights of 72,000 and 65,000, but only one major band (with a molecular weight of approximately 140,000) after electrophoresis under nondenaturing conditions. The protein inhibits the hydrolysis of single-stranded DNA by KB DNase, DNase I, DNase II, and nuclease S1, but has no effect on the hydrolysis of double-stranded DNA by these enzymes. The inhibitor causes a reduction in the rate of hydrolysis of DNA by the deoxyribonuclease, probably by reducing the effective concentration of substrate.     

Adenovirus deoxyribonucleic acid replication. II. Synthesis of viral deoxyribonucleic acid in vitro by a nuclear membrane fraction from infected KB cells.

A cell-free system for the study of viral DNA replications was developed by the isolation of a nuclear membrane fraction "DNA replication complex" from adenovirus 2-infected human KB cells late after infection. This complex which possesses both DNA polymerase activity and a virus-specific endonuclease synthesizes exclusively virus-specific DNA sequences in vitro by a semiconservative mechanism. Analysis by rate zonal sedimentation in alkaline sucrose gradients showed that the products of the reaction are small DNA chains approximately 6 to 9 S, presumably "Okazaki intermediates," that are not sealed under our in vitro conditions. Analysis by rate zonal sedimentation in neutral sucrose gradients showed that labeled viral DNA fragments are hydrogen bonded to viral 18 S DNA segments, 0.25 the size of the linear, viral 31 S DNA genome. The 18 S DNA is probably a specific cleavage product of the viral endonuclease found in the replication complex and could represent intermediates in viral DNA replication or degradation products.     

Novikoff hepatoma deoxyribonucleic acid polymerase. Purification and properties of a homogeneous beta polymerase.

Deoxyribonucleic acid polymerase-beta (EC 2.7.7.7) FROM THE Novikoff hepatoma has been purified over 200 000-fold (based on the increase in specific activity), by ammonium sulfate fractionation and chromatography on DEAE-Sephadex, phosphocellulose, hydroxylapatite, and DNA-cellulose. The enzyme is remarkably stable through all stages of purification until DNA-cellulose chromatography when it must be kept in buffers containing 0.5 M NaCl and 1 mg/ml bovine serum albumin for stability. The enzyme appears to be homogeneous as evidenced by a single stainable band when subjected to electrophoresis in polyacrylamide gels of different porosity. The stainable band corresponds to the DNA polymerase as determined by slicing sister gels and assaying for enzyme activity. The specific activity of the homogeneous preparation is about 60 000 units/mg. The enzyme lacks detectable exonuclease or endonuclease activity. It has a molecular weight of 32 000 as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. In sucrose gradients, the molecular weight is estimated at 31 000. The isoelectric point of the hydroxylapatite fraction enzyme is 8.5. The Novikoff beta-polymerase requires all four deoxyribonucleoside triphosphates, primer-template, and a divalent cation for maximal activity. The apparent Km for total deoxyribonucleoside triphosphate is 7-8 muM and for DNA 125 mug/ml. Activated DNA, rendered 7% acid soluble by DNase I, is the preferred primer-template, although a number of synthetic polynucleotides can by efficiently utilized, particularly in the presence of Mm2+ optimum is 7 mM; the Mn2+ optimum is 1 mM. The pH optimum is 8.4 in Tris-HCl or 9.2 in glycine buffer. The beta-polymerase is sstimulated about twofold by NaCl or KCl at an optimum of 50-100 MM, and the enzyme maintains considerable activity at high ionic strengths. The DNA polymerase is inhibited by ethanol, acetone, and a variety of known polymerase inhibitors. Glycols stimulate the enzyme as does spermine or spermidine. Unlike most beta-polymerases, the Novikoff enzyme is moderately sensitive to N-ethylmaleimide.