A high molecular weight DNA polymerase from Drosophila melanogaster embryos. Purification, structure, and partial characterization.

The DNA polymerase of early embryos of Drosophila melanogaster has been purified to near-homogeneity. The purified enzyme gave a single, catalytically active protein band after polyacrylamide gel electrophoresis, under nondenaturing conditions. Four polypeptides with molecular weights 43,000, 46,000, 58,000, and 148,000 were resolved when this band was electrophoresed under denaturing conditions. At high ionic strengths, the DNA polymerase had a sedimentation coefficient of 8.7 S, a Stokes radius of 78 A and frictional ratio of 1.81, parameters that yield a molecular weight of 280,000. The purified DNA polymerase possessed no detectable endo- or exodeoxyribonuclease, ATPase, or RNA polymerase activity. Using an "activated" DNA template-primer, the enzyme had a pH optimum of 8.5. It was stimulated by (NH4)2SO4, KCl, and to a lesser extent, NaCl. A divalent metal cation was absolutely required; MgCl2 stimulating activity 7-fold more than MnCl2. It was inhibited by low concentrations of N-ethylmaleimide and Aphidicolon. Thus the DNA polymerase of D. melanogaster resembles most closely the alpha-DNA polymerases that have been purified from mammalian cells.     

Immunoaffinity-purified DNA polymerase alpha from a mouse temperature-sensitive mutant, tsFT20 strain, is heat-labile

We have purified DNA polymerase alpha from a temperature-sensitive mutant cell line of mouse FM3A cells, tsFT20, that shows temperature-sensitive activity of DNA polymerase alpha (Murakami, Y., Yasuda, H., Miyazawa, H., Hanaoka, F., and Yamada, M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1761-1765). The purified enzyme was composed of two polypeptides with the same apparent molecular weights as those of purified DNA polymerase alpha from the parental strain, FM3A (Mr 180,000 and 68,000). Heat inactivation experiments revealed that this purified DNA polymerase alpha from tsFT20 cells was more heat-labile than the wild-type enzyme. We have also purified primase from both ts-FT20 cells and wild-type cells. Both primase fractions consist of two polypeptides with molecular weights of 54,000 and 46,000. No difference was observed between the heat labilities of the primases from tsFT20 cells and wild-type cells. Comparisons of wild-type and mutant polymerase indicated that the temperature-sensitive mutation in DNA polymerase alpha from tsFT20 cells affect the dCTP-binding site of the enzyme. The mutation also changed the optimum pH and the optimum KCl concentration of the enzyme.     

Further studies on calf thymus DNA polymerase delta purified to homogeneity by a new procedure

DNA polymerase delta from calf thymus has been purified to apparent homogeneity by a new procedure which utilizes hydrophobic interaction chromatography with phenyl-Sepharose at an early step to separate most of the calcium-dependent protease activity from DNA polymerase delta and alpha. The purified enzyme migrates as a single protein band on polyacrylamide gel electrophoresis under nondenaturing conditions. The sedimentation coefficient of the enzyme is 7.9 S, and the Stokes radius is 53 A. A molecular weight of 173K has been calculated for the native enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the homogeneous enzyme reveals two polypeptides of 125 and 48 kDa. This subunit structure differs from that of DNA polymerase delta prepared by our previous procedure, which was composed of subunits of 60 and 49 kDa [Lee, M. Y. W. T., Tan, C.-K., Downey , K. M., & So, A. G. (1981) Prog . Nucleic Acid Res. Mol. Biol. 26, 83-96], suggesting that the 60-kDa polypeptide may have been derived from the 125-kDa polypeptide during enzyme purification, possibly as the result of cleavage of an unusually sensitive peptide bond. DNA polymerase delta is separated from DNA polymerase alpha by hydrophobic interaction chromatography on phenyl-Sepharose; DNA polymerase delta is eluted at pH 7.2 and DNA polymerase alpha at pH 8.5. DNA polymerase delta can also be separated from DNA polymerase alpha by chromatography on hydroxylapatite; DNA polymerase alpha binds to hydroxylapatite in the presence of 0.5 M KCl, whereas DNA polymerase delta is eluted at 90 mM KCl.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Studies of Schizosaccharomyces pombe DNA polymerase alpha at different stages of the cell cycle.

The status of Schizosaccharomyces pombe (fission yeast) DNA polymerase alpha was investigated at different stages of the cell cycle. S.pombe DNA polymerase alpha is a phosphoprotein, with serine being the exclusive phosphoamino acid. By in vivo pulse labeling experiments DNA polymerase alpha was found to be phosphorylated to a 3-fold higher level in late S phase cells compared with cells in the G2 and M phases, but the steady-state level of phosphorylation did not vary significantly during the cell cycle. Tryptic phosphopeptide mapping demonstrated that the phosphorylation sites of DNA polymerase alpha from late S phase cells were not the same as that from G2/M phase cells. DNA polymerase alpha partially purified from G1/S cells had a different mobility in native gels from that from G2/M phase cells. The partially purified polymerase alpha from G1/S phase cells had a higher affinity for single-stranded DNA than that from G2/M phase cells. Despite the apparent differences in cell cycle-dependent phosphorylation, mobility in native gels and affinity for DNA, the in vitro enzymatic activity of the partially purified DNA polymerase alpha did not appear to vary during the cell cycle. The possible biological significance of these cell cycle-dependent characteristics of DNA polymerase alpha is discussed.     

High molecular weight deoxyribonucleic acid polymerase from crown gall tumor cells of periwinkle (Vinca rosea).

A high molecular weight (6 S) plant DNA polymerase from axenic Vinca rosea tissue culture cells has been purified 2200-fold and characterized. The enzyme has a molecular weight of 105 000 (+/-5000). Sodium dodecyl sulfate-acrylamide gel electrophoresis of the purified enzyme yields polypeptide subunits having molecular weights of 70 000 and 34 000. The purified enzyme has a pH optimum of 7.5; a cation requirement optimum of 6 mM Mg2+ or 0.5 mM Mn2+; an apparent requirement for Zn2+; a Km of 1 muM for dTTP; and a 3.5-fold stimulation by 50 mM KCl. The enzyme is sensitive to N-ethylmaleimide (1 mM), heparin (0.1 muM), ethanol (5%), pyrophosphate (0.05 muM), and o-phenanthroline (0.1 mM) but is insensitive to rifamycin. Denatured DNA is found to be the best natural template, and only negligible activity can be demonstrated with the ribopolymer templates poly(dT)n-poly(rA)n and p(dT)10-poly(rA)n. In addition to the polymerization reaction, the enzyme catalyzes a pyrophosphate exchange reaction. Antibody to calf thymus 6-8S DNA polymerase does not inhibit DNA polymerase from Vinca rosea, suggesting no antigenic relationships between the mammalian and plant enzymes.     

A mitochondrial DNA polymerase from embryos of Drosophila melanogaster. Purification, subunit structure, and partial characterization

The mitochondrial DNA polymerase has been purified to near-homogeneity from early embryos of Drosophila melanogaster. Sodium dodecyl sulfate gel electrophoresis of the highly purified enzyme reveals two polypeptides with molecular masses of 125,000 and 35,000 daltons, in a ratio of 1:1. The enzyme has a sedimentation coefficient of 7.6 S and a Stokes radius of 51 A. Taken together, the data suggest that the D. melanogaster DNA polymerase gamma is a heterodimer. DNA polymerase activity gel analysis has allowed the assignment of the DNA polymerization function to the large subunit. The DNA polymerase exhibits a remarkable ability to utilize efficiently a variety of template-primers including gapped DNA, poly(rA).oligo(dT) and singly primed phi X174 DNA. Both the crude and the highly purified enzymes are stimulated by KCl, and inhibited by dideoxythymidine triphosphate and by N-ethylmaleimide. Thus, the catalytic properties of the near-homogeneous Drosophila enzyme are consistent with those of DNA polymerase gamma as partially purified from several vertebrates.     

Purification and characterization of two new high molecular weight forms of DNA polymerase delta

Two high molecular weight DNA polymerases, which we have designated delta I and delta II, have been purified from calf thymus tissue. Using Bio Rex-70, DEAE-Sephadex A-25, and DNA affinity resin chromatography followed by sucrose gradient sedimentation, we purified DNA polymerase delta I 1400-fold to a specific activity of 10 000 nmol of nucleotide incorporated h-1 mg-1, and DNA polymerase delta II was purified 4100-fold to a final specific activity of 30 000 nmol of nucleotide incorporated h-1 mg-1. The native molecular weights of DNA polymerase delta I and DNA polymerase delta II are 240 000 and 290 000, respectively. Both enzymes have similarities to other purified delta-polymerases previously reported in their ability to degrade single-stranded DNA in a 3' to 5' direction, affinity for an AMP-hexane-agarose matrix, high activity on poly(dA) X oligo(dT) template, and relative resistance to the polymerase alpha inhibitors N2-(p-n-butylphenyl)dATP and N2-(p-n-butylphenyl)dGTP. These two forms of DNA polymerase delta also share several common features with alpha-type DNA polymerases. Both calf DNA polymerase delta I and DNA polymerase delta II are similar to calf DNA polymerase alpha in molecular weight, are inhibited by the alpha-polymerase inhibitors N-ethylmaleimide and aphidicolin, contain an active DNA-dependent RNA polymerase or primase activity, display a similar extent of processive DNA synthesis, and are stimulated by millimolar concentrations of ATP. We propose that calf DNA polymerase delta I, which also has a template specificity essentially identical with that of calf DNA polymerase alpha, could be an exonuclease-containing form of a DNA replicative enzyme.     

Human cell DNA replication is mediated by a discrete multiprotein complex

A discrete high molecular weight multiprotein complex containing DNA polymerase alpha has been identified by a native Western blotting technique. An enrichment of this complex was seen at each step in its purification. Further purification of this complex by ion-exchange chromatography indicates that the peak of DNA polymerase alpha activity co-purifies with the peak of in vitro SV40 DNA replication activity eluting from the column. The complex has a sedimentation coefficient of 18S in sucrose density gradients. We have designated this complex as the DNA synthesome. We further purified the DNA synthesome by electroeluting this complex from a native polyacrylamide gel. The eluted complex retains in vitro DNA synthetic activity, and by Western blot analysis, contains DNA polymerase delta, proliferating cell nuclear antigen, and replication protein A. Enzymatic analysis of the electroeluted DNA synthesome indicates that the synthesome contains topoisomerase I and II activities, and SDS-PAGE analysis of the electroeluted DNA synthesome revealed the presence of at least 25 major polypeptides with molecular weights ranging from 20 to 240 kDa. Taken together, our evidence suggests that the DNA synthesome may represent the minimal DNA replication unit of the human cell.     

Single-stranded-DNA-dependent ATPase from HeLa cells that stimulates DNA polymerase alpha-primase activity: purification and characterization of the ATPase

A single-stranded DNA-dependent ATPase that cofractionates during the early stages of purification of a multiprotein DNA polymerase alpha complex from HeLa cells has been purified to homogeneity. The ATPase is part of a 16S multienzyme DNA polymerase alpha complex that is fully active in SV40 DNA replication in vitro. The ATPase hydrolyzes ATP to ADP in a reaction that is completely dependent on the presence of DNA. DNA in single-stranded form is strongly preferred as a cofactor, and polydeoxynucleotides with adenine or thymidine residues are highly effective. Glycerol gradient sedimentation showed that the purified ATPase sedimented at an s20,w of 7 S, and polyacrylamide gel electrophoresis under denaturing conditions reveals two polypeptides with relative molecular weights of 83,000 and 68,000. Both of these polypeptides have purine nucleotide binding sites as revealed by photoaffinity cross-linking experiments. ATP binds to the two subunits more efficiently than GTP, and CTP or UTP does not cross-link with the two polypeptides. DNA synthesis catalyzed by purified HeLa cell DNA polymerase alpha-primase is stimulated in the presence of ATPase and ATP at an optimum concentration of 2 mM. Analysis of the DNA product by gel electrophoresis indicates that with poly(dT) but not phage M13 DNA as template the ATPase overcomes a lag and decreases the length of nascent DNA chains synthesized by the DNA polymerase alpha-primase complex.     

RNA-dependent DNA polymerase of an endogenous type C virus of mice: purification and partial characterization

An RNA-dependent DNA polymerase was isolated from purified virions of endogenous oncornaviruses released by the MOPC-315 murine myeloma cell line. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme was found to consist of two major polypeptides with molecular weights of about 28,000 and 26,500. The active enzyme had a molecular weight of approximately 56,000, as calculated from its sedimentation on glycerol density gradients, indicating that it is probably a dimer of the two subunit polypeptides. The isolated MOPC-315 virus polymerase exhibited all three activities known to be found in the DNA polymerase from oncornaviruses, namely, an RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, and an RNase H. The RNA-dependent polymerase activity showed a prounced preference for Mn2+ over Mg2+, whereas the DNA-dependent and RNase H reactions were catalyzed by these two cations to an almost equal extent. The purified polymerase was found to be immunologically related to the polymerase of Rauscher murine leukemia virus.     

The role of DNA polymerases alpha, beta and gamma in nuclear DNA synthesis.

The effects of the inhibitors 2'3' dideoxythymidine triphosphate (ddTTP) and 1-beta-D-arabinofuranosyl cytosine triphosphate (araCTP) on DNA synthesis in isolated S-phase HeLa S3 nuclei have been examined. These effects are compared with the effects of the same inhibitors in partially purified preparations of DNA polymerases alpha and beta. The effect of ddTTP on partially purified DNA polymerase gamma was also tested. DNA polymerases beta and gamma were very sensitive to ddTTP whereas DNA polymerase alpha and DNA synthesis in isolated nuclei were quite resistant. The synthesis and subsequent ligation of primary DNA pieces ('Okazaki fragments') were not affected by the presence of this inhibitor. DNA synthesis in isolated nuclei and DNA polymerase alpha activity were very sensitive to araCTP whereas DNA polymerase beta was almost totally resistant to the inhibitor. The results indicate a major role for DNA polymerase alpha in DNA replication.     

Simultaneous purification of RNA-dependent DNA polymerase and gs-antigen from Rauscher leukemia virus.

RNA-dependent DNA polymerase and gs-antigen were purified simultaneously from Rauscher leukemia virus by sequential column chromatography on phosphocellulose. The partially purified RNA-dependent DNA polymerase has a molecular weight of 70,000 and is free of cellular DNA polymerase, deoxynucleotidyl terminal transferase, RNase and DNase. The partially purified RNA-dependent DNA polymerase can efficiently copy oligo dT·poly rA and oligo dG·poly rC. The purified gs-antigen shows a single band on SDS-polyacrylamide gel with a molecular weight of 30,000. It is active immunologically and possesses both group and interspecies activity.     

Structural study of immunoaffinity-purified DNA polymerase alpha-DNA primase complex from calf thymus

The DNA polymerase alpha-DNA primase complex was purified over 17,000-fold to near homogeneity from calf thymus using an immunoaffinity column. Sodium dodecyl sulfate gel electrophoresis revealed three polypeptides with molecular weights of 140, 50 and 47 kDa, in a ratio of 1:2:0.25. The complex showed a sedimentation coefficient of 9.7 S, a Stokes radius of 56 A and a native molecular weight of 250-260 kDa. Taken together, the data suggest that the calf thymus dNA polymerase alpha-DNA primase complex is essentially a heterotrimer of large (140 kDa) and small (50 kDa) subunits in a ratio of 1:2, with a globular conformation. Electron-microscopic studies of the complex revealed a spherical particle of 120 A in diameter, in agreement with the physiochemical results. The binding of the complex to DNA was also demonstrated.     

Deoxyribonucleic acid polymerase from Physarum polycephalum. Properties of the major cytoplasmic activity in exponentially growing microplasmodia

DNA polymerase was purified 1000-fold from the cytoplasm of microplasmodia of the myxomycete Physarum polycephalum. The activity was found in two forms exhibiting molecular weights of 204000 and 116000 respectively. Both forms eluted together from DNA-cellulose and DEAE-Sephadex columns. The Stokes radii were 6.5 and 5.5 nm. The sedimentation coefficients were 7.6 and 5.2 S. The frictional ratios of 1.69 suggest a highly hydrated and/or an asymmetric structure of the molecule. The enzyme-catalyzed reaction was sensitive to N-ethylmaleimide (60% inhibition by 1 mM). Unlike DNA polymerase alpha from mammalian cells the Physarum enzyme was stimulated by 30 mM NaCl. Activated DNA was the preferred template. Poly(A) . (DT)12 was not accepted. The Km value for deoxynucleoside triphosphates was 3 micron, for activated DNA 50 microgram/ml and for Mg2+ at the optimum [k+] of 150 mM about 0.6 mM.     

A simplified procedure for the analysis of DNA polymerase III levels in Bacillus subtilis strains.

A simple and reproducible procedure is described which allows the fast and almost quantitative removal of DNA polymerases I and II from DNA polymerase III, in crude extracts of polA+ strains of Bacillus subtilis. The procedure entails streptomycin sulfate and ammonium sulfate fractionations; subsequent analysis of the partially purified preparation by G-200 chromatography, DEAE cellulose chromatography and density gradient sedimentation, shows that the ammonium sulfate fraction contains less than 5% of the total activity as DNA polymerase I and less than 2% as DNA polymerase II. The purification procedure, up to the ammonium sulfate step, was utilized for the analysis of the level of DNA polymerase III in several B. subtilis mutants, with results comparable to those obtained from the corresponding polA- strains following more cumbersome purification procedures. The M.W. of the purified form is of 227.000, somewhat greater than the published values. The early fractions of the purification have revealed the existence of a form with a M.W. of 426.000; the nature of this form, which has been observed in several instances and which is very unstable and short-lived, is under investigation.     

Deoxyribonucleic acid poymerase of BHK-21/C13 cells. Heterogeneity, molecular asymmetry and subcellular distribution of the enzymes.

Nuclear and cytoplasmic fractions were prepared from exponentially-growing BHK-21/C13 cells; DNA polymerase was extracted from them and analysed by gel filtration and sucrose-density-gradient centrifugation. DNA polymerase I is heterogeneous comprising species covering a considerable range of molecular weights. These have been tentatively identified as four subspecies of apparent molecular weights 900000-1000000 (IA), 460000-560000 (IB), 270000-320000 (IC) and 140000-200000 (ID), as assessed by gel filtration through Sepharose 6B. DNA polymerase II has a mol.wt. of 46000 +/- 4000 as assessed by gel filtration on Sepharose 6B, and 48000 +/- 2000 as assessed by gel filtration on Sephadex G-100. Sedimentation analyses on sucrose density gradients showed that the DNA polymerase I species had sedimentation coefficients predominantly in the range 6-8 S. DNA polymerase II had predominantly a sedimentation coefficient of 3.2 S although a component with lower sedimentation coefficient was found. The lack of correlation between the molecular weights derived from gel filtration and the sedimentation coefficients is attributed to molecular asymmetry. DNA polymerase I was found to be associated predominantly with the cytoplasm although certain types of nuclear preparation contained large amounts of it. DNA polymerase II was found to be mostly if not exclusively in nuclear preparations.     

Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA

A procedure is described for the large-scale purification of light (L) and heavy (H) chain mRNAs from plasmacytomas produced in mice. Intact RNA is selectively precipitated in high yield from frozen tumors homogenized in 3 M LiCl and 6 M urea. L and H-chain mRNAs were purified by oligo(dT)-cellulose chromatography and either sucrose gradient centrifugation in conditions preventing aggregation or by means of high-resolution preparative gel electrophoresis under non-denaturing conditions. gamma 2a and alpha H-chain mRNAs sedimented as major components at 15.5 S and 16.5 S respectively, when L-chain mRNAs sedimented as 12-S species. H-chain mRNAs isolated by continuous elution during preparative gel electrophoresis were completely separated from both L-chain mRNA and residual 18-S rRNA, and migrated as single components of 1900 +/- 50 nucleotides on analytical denaturing gels. The partially purified H-chain mRNAs were translated into major components of molecular weights of 56,000 (gamma 2a) and 60,000 (alpha) in an mRNA-dependent rabbit reticulocyte lysate, whereas L-chain mRNAs yielded polypeptides of molecular weights of 25,000 (gamma) and 27,000 (chi). Up to 95% of the translation products directed by the purified mRNAs were immunoprecipitated using specific antisera. The purity of L and H-chain mRNAs was assessed by hybridization of corresponding cDNAs with excess recombinant plasmid DNA. The results indicated a minimum purity of 47% (gamma 2a), 62% (alpha), for H-chain mRNAs and 60% (chi), for L-chain mRNAs.