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.     

DNA-processing activities associated with the purified alpha, beta 2, and alpha beta molecular forms of avian sarcoma virus RNA-dependent DNA polymerase.

The RNA-dependent DNA polymerase purified from B77 avian sarcoma virus exhibited two distinct DNA-processing activities. The alpha and beta 2 isoenzymes possessed an endodeoxyribonuclease activity capable of nicking simian virus 40 superhelical DNA, whereas the alpha beta isoenzyme performed as an untwisting topoisomerase. Both activities associated with the three molecular forms of the retroviral DNA polymerase were dependent on the presence of either Mn2+ or Mg2+ ions. From analysis of the denaturated DNA products, it is apparent that the alpha and beta 2 isoenzymes introduced two nicks, one per each strand in the superhelical simian virus 40 DNA molecules, whereas the alpha beta polymerase converted these supercoiled molecules to the relaxed covalently closed circular form. The notion that the DNA-processing activities are located on the DNA polymerase molecules was supported by the following: (i) the three isoenzymes were of a high purity; (ii) the activities cosedimented in glycerol gradients with the DNA polymerase activities of the alpha, beta 2, and alpha beta molecular forms; and (iii) immunoglobulin directed against the purified polymerase immunoprecipitated the DNA-processing activities. Chemical treatments of the DNA polymerase molecules (with pyridoxalphosphate, iodoacetamide, and sulfhydryl reagents), which inhibited the polymerase activity, also suppressed the endonucleolytic and topoisomerase activities, suggesting that cystein and amino groups play an important role in the active sites of the DNA-processing activities as well.     

Comparative Study of Recombinant Rat Nucleoside Diphosphate Kinases α and β By Intrinsic Protein Fluorescence

Abstract

Nucleoside diphosphate (NDP) kinases of mammals are hexamers of two sorts of randomly associated highly homologous subunits of 152 residues each and, therefore exist in cell as NDP kinase isoforms. The catalytic properties and three-dimensional structures of the isoforms are very similar. The physiological meaning of the existence of the isoforms in cells remained unclear, but studying recombinant rat NDP kinases a and β, each containing only one sort of subunits, we discovered that, in contrast to the isoenzyme β, NDP kinase α is able to interact with the complex between bleached rhodopsin and G-protein transducin in retinal rod membranes at lowered pH values (Orlov et al. FEBS Lett. 389, 186–190, 1996). In order to search for possible molecular basis of such differences between these isoenzymes, a detailed comparative study of their intrinsic fluorescence properties in a large range of solvent conditions was performed in this work. The isoenzymes α and β both contain the same three tryptophan (Trp78, 133, 1nd 149) and four tyrosine (Tyr 52, 67, 147, and 151) residues per subunit, but exhibit pronounced differences in their fluorescence properties (both in spectral positions and shape and quantum yield values) and behave differently under pH titration. Whereas NDP kinase a undergoes spectral changes in the pH range 5–7 with the mid-point at 6.2, no unequivocal indication of a structural change of NDP kinase β under pH titration from 9 to 5 was obtained. Since the pH dependencies obtained for fluorescence of isoenzyme α resembles the dependence of its binding to the rhodopsin-transducin complex it was suggested that the differences between the NDP kinase isoenzymes α and β in the pH-induced behavior, revealed by the fluorescence spectroscopy, and the differences in their ability to interact with rhodopsin-transducin complex may have the same physical nature, that would be a physico-chemical reason of possible functional dissimilarity of NDP kinase isoforms in cell. An additional analysis of three-dimensional structure of homologous NDP kinases revealed that the source of the differences in fluorescence properties and pH-titration behavior between the isoenzymes α and β may be due to the difference in their global electrostatic charges, rather than to any structural differences between them at neutral pH. The unusually high positive electrostatic potential at he deeply buried active site Tyr52 makes possible that it exists in deprotonated tyrosinate form at neutral and moderately acidic solution. Such a possibility may account for rather unusual fluorescence properties of NDP kinase α: (i) rather long-wavelength emission of NDP kinase a at ca. 340 nm at pH ca. 8 at extremely low accessibility to external quenchers and, possibly, (ii) an unusually high quantum yield value (ca. 0.42).     

Dissociation of alpha beta DNA polymerase of avian myeloblastosis virus by dimethyl sulfoxide.

The alpha beta DNA polymerase of avian myeloblastosis virus was treated with dimethyl sulfoxide to dissociate the enzyme subunits. The dimethyl sulfoxide treated enzymes were passed over phosphocellulose to purify and characterize the dissociated subunits as well as to remove the dimethyl sulfoxide. RNA-directed DNA polymerase, RNase H, and nucleic acid-binding activity were monitored, as well as the subunit structure (on sodium dodecyl sulfate-polyacrylamide gels) of the various enzyme species obtained. With 30% dimethyl sulfoxide, the majority of DNA polymerase and RNase H activities as well as the alpha subunit were displaced from the alpha beta DNA polymerase position on phosphocellulose (0.23 M potassium phosphate) to the alpha DNA polymerase position (0.1 M). The association of DNA polymerase and RNase H activities with the alpha subunit suggests that alpha is the enzymatically active subunit in alpha beta. In addition to alpha DNA polymerase, a minor polymerase species eluted from phosphocellulose at 0.4 M potassium phosphate. The dissociated beta subunit eluted from phosphocellulose at a wide range of salt concentrations (0.28 to 0.5 M potassium phosphate). The dissociated beta subunit bound 3H-labeled murine leukemia virus RNA and [3H]poly(dT)-poly(dA) approximately 20-fold more avidly than alpha DNA polymerase alone. In contrast to the results with the alpha subunit, there was no correlation between DNA polymerase and RNase H activity profiles and the elution profile of the beta subunit from phosphocellulose. These observations suggest the beta subunit is either enzymatically inactive or possesses limited DNA polymerase and RNase H activity when compared with the alpha subunit.     

Cross-linking of transmembrane helices in proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli: implications for the structure and function of the membrane domain

Proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an α and a β subunit of 54 and 49 kDa, respectively, and is made up of three domains. Domain I (dI) and III (dIII) are hydrophilic and contain the NAD(H)- and NADP(H)-binding sites, respectively, whereas the hydrophobic domain II (dII) contains 13 transmembrane α-helices and harbours the proton channel. Using a cysteine-free transhydrogenase, the organization of dII and helix-helix distances were investigated by the introduction of one or two cysteines in helix-helix loops on the periplasmic side. Mutants were subsequently cross-linked in the absence and presence of diamide and the bifunctional maleimide cross-linker o-PDM (6 Å), and visualized by SDS-PAGE.In the α₂β₂ tetramer, αβ cross-links were obtained with the αG476C-βS2C, αG476C-βT54C and αG476C-βS183C double mutants. Significant αα cross-links were obtained with the αG476C single mutant in the loop connecting helix 3 and 4, whereas ββ cross-links were obtained with the βS2C, βT54C and βS183C single mutants in the beginning of helix 6, the loop between helix 7 and 8 and the loop connecting helix 11 and 12, respectively. In a model based on 13 mutants, the interface between the α and β subunits in the dimer is lined along an axis formed by helices 3 and 4 from the α subunit and helices 6, 7 and 8 from the β subunit. In addition, helices 2 and 4 in the α subunit together with helices 6 and 12 in the β subunit interact with their counterparts in the α₂β₂ tetramer. Each β subunit in the α₂β₂ tetramer was concluded to contain a proton channel composed of the highly conserved helices 9, 10, 13 and 14.     

Enolase isoenzymes as markers of differentiation in teratocarcinoma cells and normal tissues of mouse

The changing profile of enolase (EC 4.2.1.11) isoenzymes in differentiating mouse cells has been traced by the use of specific antisera to the three subunits α, β, and γ. The amounts of the isoenzymes were measured in a variety of tissues during normal mouse development and during the differentiation of mouse teratocarcinoma cells in culture and as tumors. One isoenzyme is predominant in the early cells of the developing mouse embryo, namely, the homodimer made up of α subunits. The same isoenzyme is also the sole form detected in undifferentiated teratocarcinoma (embryonal carcinoma) cells. The isoenzyme form remains unchanged in developing primitive and definitive endoderm of the embryo. Similarly, endoderm cells formed by differentiation of embryonal carcinoma cells contained only αα enolase. In contrast, during the development of striated muscle and of brain, increasing proportions of β and γ subunits, respectively, were detected. Thus enolase was found to be a marker of the differentiation of these tissues. This conclusion was substantiated by finding significant amounts of the β subunit in teratocarcinoma cell cultures which had formed beating striated muscle in vitro.     

Phosphorylation of the GABAa/Benzodiazepine Receptor α Subunit by a Receptor-Associated Protein Kinase

Abstract: Partially purified preparations of GABA_a/benzodiazepine receptor from rat brain were found to contain high levels of a protein kinase activity that phosphorylated a small number of proteins in the receptor preparations, including a 50-kilodalton (kD) phosphoprotein that comigrated on two-dimensional electrophoresis with purified, immunolabeled, and photolabeled receptor α subunit. Further evidence that the comigrating 50-kD phosphoprotein was, in fact, the receptor α subunit was obtained by peptide mapping analysis: the 50-kD phosphoprotein yielded one-dimensional peptide maps identical to those obtained from iodinated, purified α subunit. Phosphoamino acid analysis revealed that the receptor α subunit is phosphorylated on serine residues by the protein kinase activity present in receptor preparations. Preliminary characterization of the receptor-associated protein kinase activity suggested that it may be a second messenger-independent protein kinase. Protein kinase activity was unaltered by cyclic AMP, cyclic GMP, calcium plus calmodulin, calcium plus phosphatidylserine, and various inhibitors of these protein kinases. Examination of the substrate specificity of the receptor-associated protein kinase indicated that the enzyme preferred basic proteins as substrates. Endogenous phosphorylation experiments indicated that the receptor α subunit may also be phosphorylated in crude membranes by a protein kinase activity present in those membranes. As with phosphorylation of the receptor in purified preparations, its phosphorylation in crude membranes also appeared to be unaffected by activators and inhibitors of second messenger-dependent protein kinases. These findings raise the possibility that the phosphorylation of the α subunit of the GABA_a/ benzodiazepine receptor by a receptor-associated protein kinase plays a role in modulating the physiological activity of the receptor in vivo.     

Control of DNA polymerase α, β and γ activities in heat- and cold-sensitive mammalian cell-cycle mutants

Two heat-sensitive (arrested in G₁ at 39.5°C) and two cold-sensitive (arrested in G₁ at 33°C) clonal cell-cycle mutants of the murine P-815-X2 mastocytoma line were tested for DNA polymerase α, β and γ activities. After transfer of mutant cells to the respective nonpermissive temperature, DNA polymerase α activities decreased more slowly than relative numbers of cells in S phase. Furthermore, numbers of DNA-synthesizing cells decreased to near-zero levels, whereas polymerase α activities in arrested cells were as high as 15–40% of control values. After return of arrested cells to the permissive temperature, polymerase α activities increased essentially in parallel with relative numbers of cells in S phase. In contrast to the changes in thymidine kinase (Schneider, E., Müller, B. and Schindler, R. (1983) Biochim. Biophys. Acta 741, 77–85), the decrease of polymerase α during entry of cells into proliferative quiescence thus appears to be under rather relaxed control, while after return of arrested cells to the permissive temperature the increase in polymerase α is tightly coupled with reentry of cells into S phase. For DNA polymerase β and γ activities, no obvious correlation with changes in the proliferative state of cells was detected.     

Arabinosyl nucleosides. XIII. Influence of arabinofuranosylthymine on DNA-, RNA- and protein-synthesizing systems in vitro

The natural metabolite of the sponge Cryptotethya crypta, arabinofuranosylthymine (araThd), is intracellularly phosphorylated to araTTP. The present study demonstrates that araTTP inhibits both isolated DNA polymerases α and the DNA polymerase β from L5178y cells competitively with respect to the analogous substrate dTTP. The affinity of araTTP is higher to the DNA polymerase α than to the DNA polymerase β.The activity of mammalian DNA-dependent RNA polymerases I, II and III as well as the incorporation rate of a protein cellfree system is not affected by high doses of araTTP.     

Differential inactivation of DNA polymerases α and β by aldehyde compounds

The effects of cyclohexanecarboxaldehyde, benzaldehyde and protocatechualdehyde on the activities of DNA polymerases α, β and E. coli DNA polymerase I were investigated. On direct addition of the aldehydes to the DNA polymerase assay mixture containing activated DNA or poly(dA) (dT)_12–18 as a template, DNA polymerase α was most strongly inhibited by the aldehyde compounds, while DNA polymerases β and I were resistant to such aldehyde inhibition. On preincubation of the enzymes with aldehyde, both DNA polymerases α and β were inactivated; however, DNA polymerase β was protected from the inactivation when activated DNA was added to the preincubation mixture. The inhibition of DNA polymerase α by aldehyde was noncompetitive with regard to the substrate dNTP and competitive with regard to the template DNA. The extent of inhibition of DNA polymerase α by aldehyde was partly reduced by the addition of cysteine to the reaction mixture.     

Rifamycin derivatives: specific inhibitors of nucleic acid polymerases

Rifampicin and three rifamycin SV derivatives with different lipophilic side chains were tested as inhibitors of a number of purified enzymes including the α and αβ forms of RNA-directed DNA polymerase of avian myeloblastosis virus (AMV). $\text{AF}\text{ABDMP}$ (2,5-dimethyl-4-N-benzyl demethyl rifampicin), $\text{AF}\text{013}$ (O-n-octyloxime of 3-formyl rifamycin SV) and C-27 (rifamycin SV with a dicyclohexylalkyl substituted piperidyl ring at the 3-position) at concentrations less than 20 to 40 μg/ml completely inhibited the RNA- and DNA-directed DNA polymerase and RNase H activities of both AMV enzymes. Rifampicin was inactive at 100 μg/ml. When used against a variety of non-polymerizing enzymes such as alkaline phosphatase, glutamate-oxaloacetate transaminase, DNase I, and RNase A, these derivatives were inactive at drug concentrations between 100 and 200 μg/ml. Polynucleotide phosphorylase was inhibited slightly by all three derivatives. These results support the idea that rifamycin SV derivatives with appropriate 3-substituted side-chains are specific inhibitors of nucleic acid polymerizing enzymes.     

Hepatitis B virus: DNA polymerase activity of deletion mutants

The hepadnavirus P gene product is a multifunctional protein with priming, DNA- and RNA-dependent DNA polymerase, and RNase H activities. Nested N- or C-terminal deletion mutations and deletions of domain(s) in human HBV polymerase have been made. Wild-type and deletion forms of MBP-fused HBV polymerase were expressed in E. coli, purified by amylose column chromatography, and the DNA-dependent DNA polymerase activities of the purified proteins were compared. Deletion of the terminal protein or spacer regions reduced enzyme activity to 70%, respectively. However, deletion of the RNase H domain affected polymerase activity more than that of the terminal protein or spacer region. The polymerase domain alone or the N-terminal deletion of the polymerase domain still exhibited enzymatic activity. In this report, it is demonstrated that the minimal domain for the polymerizing activity of the HBV polymerase is smaller than the polymerase domain.