Purification and characterization of varicella-zoster virus-induced DNA polymerase.

Infection of WI-38 human fibroblasts with varicella-zoster virus led to the stimulation of host cell DNA polymerase synthesis and induction of a new virus-specific DNA polymerase. This virus-induced DNA polymerase was partially purified and separated from host cell enzymes by DEAE-cellulose and phosphocellulose column chromatographies. This virus-induced enzyme could be distinguished from host cell enzyme by its chromatographic behavior, template specificity, and its requirement of salt for maximal activity. The enzyme could efficiently use poly(dC).oligo(dG)12-18 as well as poly(dA).oligo(dT)12-18 as template-primers. It required Mg2+ for maximal polymerization activity and was sensitive to phosphonoacetic acid, to which host alpha- and beta-DNA polymerase were relatively resistant. In addition, this induced DNA polymerase activity was enhanced by adding 60 mM (NH4)2SO4 to the reaction mixture.     

Intracisternal A particles from FLOPC-1 BALB/c myeloma: presence of high-molecular-weight RNA and RNA-dependent DNA polymerase.

Intracisternal A particles from the FLOPC-1 line of BALB/c myeloma have been shown to contain high-molecular-weight RNA (60 to 70S) that is sensitive to RNase, alkali degradation, and heat but resistant to Pronase treatment. The intracisternal A-particle RNA contains tract of poly (A) approximately 180 nucleotides long. As shown in a reconstitution experiment, by antigenic analysis of A-particle preparation and the SC cytopathogenicity assay, the 70S RNA was not due to contamination by type C virus particles. The FLOPC-1 intracisternal A particles also possess an endogenous RNA-dependent DNA polymerase. The enzyme required Mn2+ or Mg2+, dithiothreitol, detergent, and four deoxyribonucleoside triphosphates for maximum activity. Enzymatic activity was maximally stimuated by poly (rC)-oligo (dG)12-18 and less with poly (rG)-oligo (dC)10 or poly (rA)-oligo (dT)12-18 as compare with synthetic DNA/DNA duplex templates such as poly (dA)-oligo (dT)12-18. The enzyme can utilize the A-particle endogenous RNA as template as shown by analysis of the early and late DNA products of the endogenous reaction by CsSO4 isopycnic gradient centrifuation and hybridization of purified 70S or 35S A-particle RNA with the purified complementary DNA product. Approximately 50% of the A-particle complementary DNA also hybridized with oncornavirus RNA.     

Separation and partial characterization of two deoxyribonucleic acid polymerases from Spiroplasma citri.

The separation and partial characterization of two deoxyribonucleic acid polymerases from Spiroplasma citri have been achieved. The two enzymes had different elution properties on diethylaminoethyl (DEAE) cellulose and differed in their sensitivity to N-ethylmaleimide (NEM), preference for different template-primers, and sedimentation velocity in linear glycerol gradients. The first enzyme activity, ScA, was retained on DEAE-cellulose and was not inhibited by NEM. Activated deoxyribonucleic acid and poly(dA)-oligo(dT12) were the preferred template-primers. Arabinosyl-cytidine triphosphate had no effect. The sedimentation coefficient of ScA was 6.3s. The second activity, ScB, was not retained on DEAE-cellulose and was inhibited by NEM. Poly(dA)-oligo(dT12) was the preferred template-primer, whereas activated DNA was only poorly utilized. ScB was not affected by arabinosyl-cytidine triphosphate, and its sedimentation coefficient was 4.4s. The polymerization activities of the two enzymes were maximum at 37 to 40 degrees C.     

DNA polymerase-beta from the nuclear fraction of sea urchin embryos: characterization of the purified enzyme

Approximately 2,500-fold purifications of DNA polymerase-beta from the nuclear fraction of blastulae of the sea urchin, Hemicentrotus pulcherrimus, was performed. The enzyme preparation, which was devoid of DNase and terminal deoxynucleotidyl transferase as contaminants, showed a sedimentation constant of 3.0 S in a sucrose density gradient, a molecular weight of 50,000 by gel filtration, and an isoelectric point of pH 8.1. The enzyme activity was resistant to sulfhydryl group inhibitors. Its optimal pH was 9.0-9.5 in Tris-maleate buffer and 10.0 in glycine buffer. The optimal NaCl concentration for the activity was 30-60 mM and about half of the activity remained at 0.4 M NaCl. As a template-primer, the enzyme preferred synthetic homopolymers to activated DNA. The order of this preference was as follows; poly (dA)-oligo (dT)12-18 greater than poly (rA)-oligo (dT)12-18 greater than activated DNA. The above results indicate that the enzyme corresponds to DNA polymerase-beta from vertebrate cells.     

Identity of DNA polymerase gamma from synaptosomal mitochondria and rat-brain nuclei

DNA polymerase gamma and mitochondrial DNA polymerase were isolated from brain nuclei and synaptosomes respectively. The presence of a single DNA polymerase in synaptosomal mitochondria was established by chromatography on DEAE-cellulose, phosphocellulose and DNA-cellulose, as well as by sedimentation analysis and isoelectric focusing. A great similarity between the purified nuclear DNA polymerase gamma and the mitochondrial enzyme was found by the following criteria: chromatographic behaviour in three column systems; essentially complete inhibition by N-ethyl-maleimide (2 mM); optimal requirements of Mn2+ (0.1 mM), Mg2+ (5 mM) and pH (8.0); template preferences, poly(A) - (dT)20-25 larger than activated DNA larger than poly(dA) - (dT)12-18; lack of activity on single-stranded polynucleotides and (dT)12-primed mRNA; molecular weight (180000), sedimentation (9.2 S) and isoelectric point (pI 5.4). We therefore conclude that brain nuclear DNA polymerase gamma and synaptosomal mitochondrial DNA polymerase are closely related and may even be identical.     

Identification of gamma-like DNA polymerase from sea urchin embryos.

A gamma-like DNA polymerase devoid of DNA polymerase-alpha and -beta activities was prepared from the nuclear fraction of blastulae of the sea urchin, Hemicentrotus pulcherrimus. The enzyme sedimented at the position of an approximate sedimentation coefficient of 3.3 S under high salt conditions by sucrose gradient centrifugation. An isoelectric point was determined to be pH 5.8. The enzyme activity was sensitive to sulfhydryl blocking reagents. Poly(rA) . oligo(dT)12--18 followed by poly(dA) . oligo(dT)12--18 was effectively utilized as a template-primer. From the above results, this polymerase seems to resemble the vertebrate DNA polymerase-gamma.     

A method of isolation and properties of DNA-dependent DNA-polymerase epsilon from human placenta]

DNA polymerase epsilon was purified to near homogeneity from human placenta. The enzyme has one subunit (170 kDa, sedimentation coefficient 8.2S), intrinsic 3'-5'-exonuclease activity, it is independent on PCNA and high processivity on poly(dA)-oligo(dT) template-primer without PCNA. It was shown, that the enzyme incorporates 3'-amino-2',3'-dideoxythymidine 5'-triphosphate in DNA, after that synthesis is stopped. Simultaneously DNA polymerase alpha was purified.     

Three forms of DNA polymerase from Drosophila melanogaster embryos. Purification and properties.

DNA polymerase was purified from Drosophila melanogaster embryos by a combination of phosphocellulose adsorption, Sepharose 6B gel filtration, and DEAE-cellulose chromatography. Three enzyme forms, designated enzymes I, II, and III, were separated by differential elution from DEAE-cellulose and were further purified by glycerol gradient centrifugation. Purification was monitored with two synthetic primer-templates, poly(dA) . (dT)-16 and poly(rA) . (dT)-16. At the final step of purification, enzymes I, II, and III were purified approximately 1700-fold, 2000-fold and 1000-fold, respectively, on the basis of their activities with poly(dA) . (dT)-16. The DNA polymerase eluted heterogeneously as anomalously high-molecular-weight molecules from Sepharose 6B gel filtration columns. On DEAE-cellulose chromatography enzymes I and II eluted as distinct peaks and enzyme III eluted heterogeneously. On glycerol velocity gradients enzyme I sedimented at 5.5-7.3 S, enzyme II sedimented at 7.3-8.3 S, and enzyme III sedimented at 7.3-9.0 S. All enzymes were active with both synthetic primer-templates, except the 9.0 S component of enzyme III, which was inactive with poly(rA) . (dT)-16. Non-denaturing polyacrylamide gel electrophoresis did not separate poly(dA) . (dT)-16 activity from poly(rA) . (dT)-16 activity. The DNA polymerase preferred poly(dA) . (dT)-16 (with Mg2+) as a primer-template, although it was also active with poly(rA) . (dT)-16 (with Mn2+), and it preferred activated calf thymus DNA to native or heat-denatured calf thymus DNA. All three primer-template activities were inhibited by N-ethylmaleimide. Enzyme activity with activated DNA and poly(dA) . (dT)-16 was inhibited by K+ and activity with poly(rA) . (dT)-16 was stimulated by K+ and by spermidine. The optimum pH for enzyme activity with the synthetic primer-templates was 8.5. The DNA polymerases did not exhibit deoxyribonuclease or ATPase activities. The results of this study suggest that the forms of DNA polymerase from Drosophila embryos have physical properties similar to those of DNA polymerase-alpha and enzymatic properties similar to those of all three vertebrate DNA polymerases.     

Biochemical and functional comparison of DNA polymerases alpha, delta, and epsilon from calf thymus.

DNA polymerase alpha, delta and epsilon can be isolated simultaneously from calf thymus. DNA polymerase delta was purified to apparent homogeneity by a four-column procedure including DEAE-Sephacel, phenyl-Sepharose, phosphocellulose, and hydroxylapatite, yielding two polypeptides of 125 and 48 kDa, respectively. On hydroxylapatite DNA polymerase delta can completely be separated from DNA polymerase epsilon. By KCl DNA polymerase delta is eluted first, while addition of potassium phosphate elutes DNA polymerase epsilon. DNA polymerases delta and epsilon could be distinguished from DNA polymerase alpha by their (i) resistance to the monoclonal antibody SJK 132-20, (ii) relative resistance to N2-[p-(n-butyl)phenyl]-2-deoxyguanosine triphosphate and 2-[p-(n-butyl)anilino]-2-deoxyadenosine triphosphate, (iii) presence of a 3'----5' exonuclease, (iv) polypeptide composition, (v) template requirements, (vi) processivities on the homopolymer poly(dA)/oligo(dT12-18), and (vii) lack of primase. The following differences of DNA polymerase delta to DNA polymerase epsilon were evident: (i) the independence of DNA polymerase epsilon to proliferating cell nuclear antigen for processivity, (ii) utilization of deoxy- and ribonucleotide primers, (iii) template requirements in the absence of proliferating cell nuclear antigen, (iv) mode of elution from hydroxylapatite, and (v) sensitivity to d2TTP and to dimethyl sulfoxide. Both enzymes contain a 3'----5' exonuclease, but are devoid of endonuclease, RNase H, DNA helicase, DNA dependent ATPase, DNA primase, and poly(ADP-ribose) polymerase. DNA polymerase delta is 100-150 fold dependent on proliferating cell nuclear antigen for activity and processivity on poly(dA)/oligo(dT12-18) at base ratios between 1:1 to 100:1. The activity of DNA polymerase delta requires an acidic pH of 6.5 and is also found on poly(dT)/oligo(dA12-18) and on poly(dT)/oligo(A12-18) but not on 10 other templates tested. All three DNA polymerases can be classified according to the revised nomenclature for eukaryotic DNA polymerases (Burgers, P.M. J., Bambara, R. A., Campbell, J. L., Chang, L. M. S., Downey, K. M., Hübscher, U., Lee, M. Y. W. T., Linn, S. M., So, A. G., and Spadari, S. (1990) Eur. J. Biochem. 191, 617-618).     

RNA-dependent DNA polymerase from a cell line derived from the bone marrow of a patient with polycythemia vera.

A RNA-dependent DNA polymerase was isolated from a human cell line derived from the bone marrow of a patient with polycythemia vera. The purification procedure included chromatography on phosphocellulose and oligo(dT)-cellulose, and glycerol gradient centrifugation. The enzyme could be distinguished from polymerase A by salt elution from phosphocellulose, utilization of poly(rC) - oligo(dG) and its molecular size of about 70000, as determined by centrifugation. Throughout the purification procedure ribonuclease H activity was co-purified. Upon dodecylsulfate-polyacrylamide electrophoresis on microgradient gels two main bands with molecular weights of 68000 and 66000 and three minor bands were detected. The enzyme preferentially used poly(rA) - oligo(dT) as template-primer compared with poly(dA) - oligo(dT). It incorporated dGMP into polymer on poly(rC) - oligo(dG).     

The mode of inhibitory action by pyridoxal 5-phosphate on DNA polymerase-alpha and -beta.

The kinetics of the inhibition of DNA polymerases-alpha and -beta from sea urchin embryos by pyridoxal 5-phosphate were studied. The inhibition of DNA polymerase-alpha activity by pyridoxal 5-phosphate was competitive with activated DNA but noncompetitive with each deoxynucleoside triphosphate. With poly(dC)-oligo(dG)12-18 as a template-primer, however, the inhibition of DNA polymerase-alpha was competitive with dGTP but noncompetitive with the template-primer. These results suggest that DNA polymerase-alpha interacts with activated DNA and poly(dC)-oligo(dG)12-18 in different ways. The inhibition of DNA polymerase-beta by pyridoxal 5-phosphate was competitive with deoxynucleoside triphosphate using activated DNA as a template-primer and noncompetitive with activated DNA. Using poly(rA)-oligo(dT)12-18 as a template-primer, DNA polymerase-beta activity yielded sigmoid curves against both dTTP and the template-primer concentrations and was inhibited by pyridoxal 5-phosphate noncompetitively with respect to both dTTP and the template-primer. These results indicate that the inhibitory mode of DNA polymerase-alpha by pyridoxal 5-phosphate is different from that of DNA polymerase-beta.     

AraUTP-Affi-Gel 10: a novel affinity absorbent for the specific purification of DNA polymerase alpha-primase

For the specific purification of eukaryotic DNA-dependent DNA polymerase alpha, we prepared two novel affinity resins bearing 5-(E)-(4-aminostyryl) araUTP as a ligand. One of them was araUTP-Sepharose 4B which was coupled directly with the ligand and the other was araUTP-Affi-Gel 10 which was coupled with the ligand through a spacer. No DNA polymerase alpha-primase activity from cherry salmon (Oncorhynchus masou) testes was bound on the araUTP-Sepharose 4B in all cases examined. On the other hand, the araUTP-Affi-Gel 10 retains this enzyme activity when poly(dA) or poly(dA)-oligo(dT)12-18 is present. The retained enzyme activity was sharply eluted around 100-mM KCl concentrations as a single peak, and this fraction showed a specific activity of about 170,000 units/mg as alpha-polymerase activity. The highly purified DNA polymerase alpha-primase isolated using the araUTP-Affi-Gel 10 contained only three polypeptides, which showed Mr values of 120,000, 62,000, and 58,000, respectively, as judged using sodium dodecyl sulfate-polyacrylamide gel electrophoresis.