The relationship between two murine DNA-dependent DNA polymerases from the cytosol and the low molecular weight DNA polymerase.

After aqueous subcellular fractionation and partial purification by phosphocellulose chromatography, murine cells are found to contain a low molecular weight DNA-dependent DNA polymerase (beta) in the nuclear fraction and two distinguishable DNA-dependent DNA polymerases (C-I and C-II) in the cytosol. Both C-I and C-II are found in testis, liver, and regenerating liver; the amount of C-I being several fold increased in the regenerating liver and in immature testis. C-I and C-II are distinguishable by the criteria of salt sensitivity, inhibition by single-stranded DNA, elution from phosphocellulose, inhibition by 0.3 mM N-ethylmaleimide, template preference, and sedimentation coefficient. C-II is dissociated by 0.25 M KC1 to an active form of DNA polymerase of sedimentation coefficient 3.5 S while C-I is not dissociated, maintaining its sedimentation coefficient of 7.2 S. Many similar chemical and physical properties of C-II and the low molecular weight nuclear DNA polymerase (beta) suggest that C-II may represent an aggregate state of beta monomers, The size, reaction properties and the increase in enzyme activity under conditions of rapid cellular proliferation suggest C-I is analogous to the alpha DNA polymerase.     

DNA polymerases of anucleated cells. Isolation and characterization of two DNA polymerases from human platelets.

Two different DNA polymerases have been purified and characterized from human platelets. In the mitochondrial fraction a unique activity of the polymerase gamma type has been found. The same enzyme is found in the extramitochondrial supernatant. A second DNA polymerase, called 'cytoplasmic' DNA polymerase has been found in the 10000 x g supernatant of human platelets. The following properties of the latter DNA polymerase from human platelets are identical to those of DNA polymerase alpha from normal cells: DEAE-cellulose and phosphocellulose chromatography, size, thermal stability, phosphonoacetic acid and ethidium bromide inhibition. However, some of its properties, like high resistance to N-ethylmaleimide and the lack of DNA polymerization using synthetic RNA primers, are those of DNA polymerase beta.     

DNA polymerases of rat liver. Partial characterization and effect of various inhibitors.

Three distinct DNA-dependent DNA polymerase activities have been partially purified from normal rat liver. Soluble activities are separable into two distinct fractions (P1 and P2) by phosphocellulose chromatography. A low-molecular-weight DNA polymerase was isolated from purified nuclei. The enzymes were characterized according to chromatographic and sedimentation behavior, enzymological properties, and response to various inhibitors. The results indicate that fraction P1 corresponds to the high-molecular-weight enzyme and suggest that polymerase P2 may be derived from partial dissociation of the high-molecular-weight enzyme. The molecular weight of polymerase P1 was estimated to be about 250 000 by Sephadex column chromatography. Both fraction P2 and nuclear DNA polymerase appeared to be low-molecular-weight enzymes. However, the molecular size of these activities was apparently different. The estimated molecular weights of nuclear and P2 enzyme are about 40 000 and 25 000, respectively. As with the nuclear enzyme, polymerase P2 (but not P1) appeared to be free of detectable exonuclease activity. All of these polymerases showed a marked preference for initiated polydeoxyribonucleotide templates. The rat liver polymerases differed in their ability to use poly[d(A-T)-A1 primer-template, as is shown by the ratios of their activity with this synthetic polymer to that with activated DNA: 0.5, 2.75, and 1.34 for P1, P2, and nuclear polymerase, respectively. Denatured DNA was a poor template for both enzymes P1 and P2, but it was inert as template for the nuclear enzyme. Although each of these polymerases required all four deoxynucleoside triphosphates for maximal activity, they catalyzed a high rate of synthesis in the absence of one or more deoxynucleoside triphosphates. Such a 'limited' synthesis was much more extensive for polymerase P2 and nuclear enzyme than for P1 was the most sensitive of the three to sulphydryl reagents, ehtidium bromide, heparin, and single-stranded DNA. The responses of P2 and nuclear enzymes to various inhibitors were very similar. However, these two enzymes respond differently to heat and high ionic strength.     

Detection of RNA-dependent DNA polymerase activity in the Xiphophorus melanoma system.

DNA polymerases have been isolated from muscle and melanoma tissues of Xiphophorus, which are similar to retroviral RNA-dependent DNA polymerases as they prefer RNA to DNA templates. They appear to associate with submicroscopic structures which exhibit a density of about 1.13 g/ml after sucrose-density-gradient centrifugation. The RNA-dependent-DNA-polymerase-like enzymes could be separated from the DNA-dependent DNA polymerases by DEAE-cellulose chromatography. Further purification on phosphocellulose revealed that the muscle enzyme eluted at the void volume and at about 0.6 M KCl, whereas most of the melanoma enzyme eluted at 0.1 M KCl. Comparison of the template primer specificities of the muscle and melanoma enzymes with those of known DNA polymerases showed obvious similarities to the RNA-dependent DNA polymerase isolated from Rous sarcoma virus.     

The deoxyribonucleic acid polymerases from the diatom Cylindrotheca fusiformis. Subcellular distribution, exonuclease activity and heterogeneity of the enzymes.

Four DNA polymerases from the marine diatom Cylindrotheca fusiformis, polymerases A, B, C and D, were further differentiated by their subcellular localization, presence of deoxyribonuclease activity, apparent heterogeneity and molecular weights. Polymerases A, B and D occur in significant amounts in the soluble fraction, suggesting that they were originally localized in the nuclei, whereas polymerase C predominates in the chloroplasts. A mitochondrial DNA polymerase was also isolated and characterized by ion-exchange chromatography. Polymerase D has an associated nuclease activity which prefers denatured DNA and Mg2+, and has a pH optimum higher than that for polymerase activity. Co-elution from a DEAE-Sephadex column and co-sedimentation in glycerol density gradients of deoxyribonuclease and polymerase D activity suggest a molecular association. Polymerases A, B and C are devoid of nuclease activity. Glycerol-gradient-sedimentation analysis showed that all DNA polymerase fractions are heterogeneous at low ionic strengths, with the appearance of a single homogeneous activity of 0.5M-KCl. Estimated molecular weights of 100000, 82000 and 120000 for polymerases A, B and C respectively were obtained from sedimentation analysis and gel filtration. Polymerase D was estimated to have a molecular weight of about 100000 as determined by sedimentation analysis alone.     

High-molecular-weight DNA polymerases from mouse myeloma. Purification and properties of three enzymes.

Cytoplasmic (high-molecular-weight) DNA polymerase was partially purified from mouse myeloma. Upon chromatography on DEAE-Sephadex, following fractionation on phosphocellulose, the enzyme was resolved into three species named CI, CII, and CIII. The species CI and CII have equal sedimentation coefficients (10.5 S) in sucrose gradients without salt. In the presence of 125 mM ammonium sulfate the sedimentation coefficients are reduced to 8.6 S. The species CIII shows sedimentation coefficients of 5.7 S and 5.2 S without salt and in the presence of 125 mM ammonium sulfate, respectively. This species is assumed to be an artifact arising from either CI or to a minor extent from CII. The optima for pH, KCl and Mg2+ concentration, and the extent of inhibition by N-ethylmaleimide are the same. However, the enzymes differ in their responses to Mn2+ (substituting for Mg-2+), and to addition of ethanol, dimethylsulfoxide, and various phospholipids in the assay mixture. The enzymes prefer poly[d(A-T - d(A-T)] or partially degraded (activated) DNA as template rather than double-stranded or single-stranded DNA. The activity on activated DNA relative to that on poly[d(A-T) - D(A-T)] was found to be 93, 66, and 29% for DNA polymerases CI, CII, and CIII, respectively.     

Purification and Characterization of DNA-dependent RNA Polymerases from Cauliflower Nuclei

DNA-dependent RNA polymerases were solubilized from nuclei of cauliflower inflorescences and purified by agarose A-1.5m, DEAE-cellulose, DEAE-Sephadex, and phosphocellulose chromatography and sucrose density gradient centrifugation. RNA polymerases I + III were separated from II by DEAE-cellulose chromatography. Subsequent chromatography on DEAE-Sephadex resolved RNA polymerase I from III. RNA polymerases I and II were further purified to high specific activity by phosphocellulose chromatography and sucrose density gradient centrifugation. RNA polymerase I was refractory to α-amanitin at 2 mg/ml. RNA polymerase II was 50% inhibited at 0.05 μg/ml, and RNA polymerase III was 50% inhibited at 1 to 2 mg/ml of α-amanitin. The enzymes were characterized with respect to divalent cation optima, ionic strength optima, and abilities to transcribe cauliflower, synthetic, and cauliflower mosaic virus DNA templates.     

Deoxyribonuclease activity found in Epstein--Barr virus producing lymphoblastoid cells.

A deoxyribonuclease activity from Epstein--Barr (EB) virus producer lymphocyte cell lines which is correlated with viral production and which is not present in virus non-producer or negative lymphocyte cell lines has been purified 220-fold with 20% recovery and characterized. This nuclease copurifies through diethylaminoethylcellulose column chromatography with the EB virus induced deoxyribonucleic acid (DNA) polymerase in EB virus producer cells which was recently reported by this laboratory, but elutes as a separate peak of activity upon phosphocellulose chromatography. This nuclease activity has a sedimentation coefficient of 4.0 S, a strong divalent cation requirement, an alkaline pH optimum, and the ability to utilize both native and denatured lymphocyte DNA as substrate, reducing both to monophosphonucleosides.     

Isolation and characterization of a DNA helicase from cytosolic extracts of calf thymus

A DNA helicase has been isolated from calf thymus tissue. The enzyme was enriched from crude cytosolic extracts by batchwise chromatography on phosphocellulose, followed by 35% ammonium sulfate precipitation, and subsequent chromatography on phenyl-Sepharose, single-stranded DNA cellulose, and AcA 44 gel filtration. The DNA helicase had a Stokes' radius of about 45? and a sedimentation coefficient of 4.3 S. The most purified fractions contained three polypeptides with apparent molecular weights of 110, 65, and 34 kDa. UV crosslinking with radioactive dATP stained all three major polypeptides. The helicase catalyzed the unwinding of a DNA primer from a single-stranded DNA template in an ATP- or dATP-dependent manner. DNA unwinding was also observed with CTP or dCTP, but with reduced efficiency. The helicase translocated from 3′ to 5′ on the single-stranded template it was bound to. Relationships between this DNA helicase and other calf thymus helicases will be discussed.     

Characterization of the deoxyribonucleic acid polymerase associated with Kilham rat virus.

Purified preparations of the parvovirus, Kilham rat virus, have associated with them a protein with DNA polymerase activity. The enzyme has been separated from the other two or three viral proteins and purified 63-fold. The viral associated enzyme was found in a single peak of DNA polymerase activity after chromatography on DEAE-cellulose, DNA-cellulose, and phosphocellulose columns. It shares some properties in common with the host cellular DNA polymerases, described in the preceding paper (Salzman, L.A., and McKerlie, L. (1975) J. Biol. Chem. 250, 5589-5595), but also has some important distinguishing characteristics. The Kilham rat virus-associated DNA polymerase has increased enzyme activity in the presence of 0.02 M KCl and has a strong preference for a synthetic DNA polymer containing deoxyadenylate and deoxythymidylate. The enzyme has a molecular weight of approximately 75,000 plus or minus 3,000 and appears to contain endonuclease activity.     

Isolation and characterization of a DNA helicase from cytosolic extracts of calf thymus

A DNA helicase has been isolated from calf thymus tissue. The enzyme was enriched from crude cytosolic extracts by batchwise chromatography on phosphocellulose, followed by 35% ammonium sulfate precipitation, and subsequent chromatography on phenyl-Sepharose, single-stranded DNA cellulose, and AcA 44 gel filtration. The DNA helicase had a Stokes' radius of about 45Å and a sedimentation coefficient of 4.3 S. The most purified fractions contained three polypeptides with apparent molecular weights of 110, 65, and 34 kDa. UV crosslinking with radioactive dATP stained all three major polypeptides. The helicase catalyzed the unwinding of a DNA primer from a single-stranded DNA template in an ATP- or dATP-dependent manner. DNA unwinding was also observed with CTP or dCTP, but with reduced efficiency. The helicase translocated from 3′ to 5′ on the single-stranded template it was bound to. Relationships between this DNA helicase and other calf thymus helicases will be discussed.     

Purification and properties of a DNA ligase from sea urchin embryos

DNA ligase was purified about 2,000-fold from blastulae of sea urchin, Hemicentrotus pulcherrimus, by means of 1 M KCl-extraction, phosphocellulose, DEAE-cellulose, Sepharose CL-6B, and double-stranded DNA cellulose column chromatography. The purified DNA ligase had a molecular weight of 80,000 (determined by Sephadex G-150) and a sedimentation coefficient of 4.1S (by glycerol gradient centrifugation). The purified enzyme required ATP and Mg2+ (or Mn2+) as cofactors for activity, and was inhibited by N-ethylmaleimide. Apparent Km values for ATP, Mg2+, and Mn2+ were 4 microM, 2.7 mM, and 0.3 mM, respectively.     

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.     

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.     

A DNA polymerase with unusual properties from the slime mold Physarum polycephalum

Two forms of a DNA polymerase have been purified from microplasmodia of Physarum polycephalum by poly(ethyleneimine) precipitation and chromatography on DEAE-Sephacel, phosphocellulose, heparin Sepharose, hydroxyapatite, DNA-agarose, blue-Sepharose. They were separated from DNA polymerase alpha on phosphocellulose and from each other on heparin-Sepharose. Form HS1 enzyme was 30-40% pure and form HS2 enzyme 60% with regard to protein contents of the preparations. Form HS2 enzyme was generated from form HS1 enzyme on prolonged standing of enzyme preparations. The DNA polymerases were obtained as complexes of a 60-kDa protein associated with either a 135-kDa (HS1) or a 110-kDa (HS2) DNA-polymerizing polypeptide in a 1:1 molar stoichiometry. The biochemical function of the 60-kDa protein remained unknown. The complexes tended to dissociate during gradient centrifugation and during partition chromatography as well as during polyacrylamide gradient gel electrophoresis under nondenaturing conditions at high dilutions of samples. Both forms existed in plasmodia extracts, their proportions depending on several factors including those which promoted proteolysis. The DNA polymerases resembled eucaryotic DNA polymerase beta by several criteria and were functionally indistinguishable from each other. It is suggested that lower eucaryotes contain repair DNA polymerases, which are similar to those of eubacteria on a molecular mass basis.     

DNA-dependent RNA polymerase from T8 Guerin tumor

DNA-dependent RNA polymerases from nuclei of T8 Guerin tumor were studied. Two enzymes were purified several hundred times by the use of ammonium sulfate precipitation, DEAE-cellulose and phosphocellulose chromatography. One of them belongs to A(I) RNA polymerases and the second to B(II) as was established from their metal and ionic strength requirements. activity in the presence of native and denatured DNA and the resistance to a-amanitin inhibition. The quantity of class A enzyme was increased compared to B, a fact observed with most neoplastic tissues so far studied. This increase of the polymerase responsible for ribosomal RNA synthesis could probably be related to malignant transformation in animals.     

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.     

Chick-embryo DNA polymerase gamma. Identity of gamma-polymerases purified from nuclei and mitochondria

The level of DNA polymerase gamma as compared to DNA polymerases alpha and beta has been determined in chick embryo by means of specific tests: the amount of gamma-polymerase in the 12-day-old chick embryo reaches about 15% of the total polymerase activity. This enzyme is mainly localized in nuclei and mitochondria, where it represents the prevailing if not the unique DNA polymerase activity. The mitochondrial DNA polymerase gamma is likely to be associated with the internal membrane or the matrix of this organelle since it is not removed by digitonin treatment. The gamma-polymerases have been purified from chick embryo nuclei and mitochondria 500-700 times by means of DEAE-cellulose, phosphocellulose and hydroxyapatite chromatographies. The purified mitochondrial DNA polymerase gamma is closely related to the homologous enzyme purified from the nuclei of the same cells. So far, they cannot be distinguished on the basis of their sedimentation, catalytical properties and response to inhibitors or denaturating agents. The purified gamma enzymes are distinct from the chick embryo DNA polymerases alpha and beta and are not inhibited by antibodies prepared against the latter enzymes. The nuclear and mitochondrial gamma-polymerases do not respond to the oncogenic RNA virus DNA polymerase assay with natural mRNAs.