Biochemical characterization of Epstein-Barr virus nuclear antigen 2A and an associated ATPase activity.

A partial purification of the Epstein-Barr-virus nuclear antigen 2A (EBNA 2A) protein from the Epstein-Barr-virus-infected lymphoblastoid cell line, Cherry, has been designed. The main purification step was immunoaffinity chromatography, based on the mAb, 115E, directed towards the carboxy terminus of EBNA 2A. This was followed by chromatography over a Blue Sepharose column. According to silver-stained SDS/PAGE, EBNA 2A was estimated to be 20% pure. The purified fractions contained an ATPase activity that was inhibited by the mAb 115E. Immunopurification of six EBNA-2A-positive cell lines and their negative counterpart showed that only fractions from EBNA-2A-positive lines contained ATPase activity. In gel-filtration experiments EBNA 2A eluted as a 75-kDa protein in conjunction with an ATPase activity. The EBNA 2A protein was covalently labeled by the ATP analog [14C]5'-[p-(fluorosulfonyl)benzoyl]adenosine. The ATPase activity was found to be optimal in the presence of 0.25 mM MgCl2 or CaCl2, whereas, in the presence of MnCl2 and ZnCl2, the activity was only about 50% of the control. High concentrations of Na2VO3 and heparin do not interfere with the activity, while 2.5 mM NaF or 0.5 M NaCl give a 50% reduction of the activity. The Km for ATP and for GTP was 13 microM and 11 microM, respectively, and the Vmax for ATP was about six-times higher than with GTP as substrate. Other low-molecular-mass non-protein phosphate esters, such as phosphoserine or phosphothreonine inhibited the ATPase activity with a Ki of 18 and 32 microM, respectively. Phosphotyrosine had a Ki of 480 microM. Serine, threonine and tyrosine had no inhibitory effect on the ATPase activity.

DNA-repair reactions by purified HeLa DNA polymerases and exonucleases.

PM2 duplex DNA substrates containing small gaps were utilized to study DNA repair reactions of extensively purified HeLa DNase V (a bidirectional double strand DNA exonuclease) and DNA polymerases beta, gamma (mitochondrial and extramitochondrial), and alpha holoenzyme, and delta as a function of ionic strength. At 50 mM NaCl, DNase V carried out extensive exonucleolytic degradation, and beta-polymerase exhibited strand displacement synthesis. However, at 150 mM NaCl, the DNase appeared only to remove damaged nucleotides from DNA termini while beta-polymerase catalyzed only gap-filling synthesis. When present in equimolar amounts, beta-polymerase and DNase V (which can be isolated as a 1:1 complex) catalyzed more degradation than synthesis at 50 mM NaCl; however, at 150 mM NaCl a coupled very limited nick translation reaction ensued. At physiological ionic strength DNA polymerase alpha holoenzyme was not active upon these substrates. In 15 mM KCl it could fill small gaps and carry out limited nick translation with undamaged DNA, but it could not create a ligatable substrate from UV-irradiated DNA incised with T4 UV endonuclease. Mitochondrial DNA polymerase gamma was more active at 150 mM NaCl than at lower ionic strengths. It readily filled small gaps but was only marginally capable of strand-displacement synthesis. The extramitochondrial form of gamma-polymerase, conversely, was less sensitive to ionic strength; it too easily filled small gaps but was not effective in catalyzing strand displacement synthesis. Finally, DNA polymerase delta was able to fill gaps of several to 20 nucleotides in 0.05 M NaCl, but at higher NaCl concentrations there was little activity. DNA polymerases delta did not demonstrate strand displacement synthesis. Therefore, at physiological ionic strength, it appears that either DNA polymerase beta or extramitochondrial DNA polymerase gamma might aid in short patch DNA repair of nuclear (or transfecting) DNAs, whereas mitochondrial gamma-polymerase might fill small gaps in mitochondrial DNA.

Inhibition of the T7 DNA polymerase by insulin.

T7 DNA polymerase reduced insulin at the same Km as thioredoxin, while the turnover number decreased. Recycling of the disulfide of thioredoxin subunit to its dithiol form was made by thioredoxin reductase. Incubation of T7 DNA polymerase with insulin decreases its ability to bind DNA and therefore inhibited polymerase and exonuclease activities. Thioredoxin reductase fully reversed this inhibition. Insulin did not induce dissociation of the T7 DNA polymerase subunits, which was tested by immunoadsorbent chromatography. No significant difference in single-stranded exonuclease compared to polymerase activity was seen in the flow through or the eluate, which had been expected if a dissociation of the subunits had occurred.

An improved purification method and a physical characterization of phage T7 DNA polymerase.

The immunoadsorbent used to purify T7 DNA polymerase contains antibodies directed towards thioredoxin. Elution of the enzyme is made by a pulse of buffer at pH 12.0. This decreases the binding capacity of the column. Binding experiments with [3H]thioredoxin showed that the effect was caused by reduction of the antibodies by thiols in alkaline buffers. T7 DNA polymerase aggregated and irreversibly lost activity in buffers of low ionic strength. Experiments with gel chromatography and glycerol density gradient centrifugation showed that 0.2 M sodium chloride was required to keep the enzyme in its monomeric form. The sedimentation coefficient and the Stokes' radius are 5.3 S and 4.6 nm respectively, evaluated by gel chromatography and glycerol density gradient centrifugation techniques. The frictional ratio of 1.49 indicates that the T7 DNA polymerase is an asymmetrical protein.

Characterization of bacteriophage T7 DNA polymerase purified to homogeneity by antithioredoxin immunoadsorbent chromatography.

DNA polymerase of bacteriophage T7 is composed of two subunits, the gene 5 protein of the phage and the host-coded thioredoxin. We have purified T7 DNA polymerase to homogeneity from T7-infected Escherichia coli B cells with a novel technique based on immunoadsorbent affinity chromatography. The enzyme binds quantitatively to a column of anti-thioredoxin Sepharose 4B and remains as an active complex in the immobilized state. It is eluted in fully active and highly purified form by a pulse of buffer at pH 12. After a final phosphocellulose chromatography, T7 DNA polymerase of better than 99% purity, as estimated from sodium dodecyl sulfate polyacrylamide gel electrophoresis, is obtained. Determination of the molecular weight by sedimentation equilibrium centrifugation gives a value of 112,000. Denaturing gels showed that the enzyme is composed of gene 5 protein (Mr = 87,000 +/- 3,000) and thioredoxin (Mr = 12,000) in a 1:1 stoichiometry. The amino acid composition of the enzyme and its spectrum was determined. The DNA polymerase activity is dependent on sulfhydryl compounds, sensitive to salt, and shows a comparatively high Km value for the four deoxyribonucleotides. The enzyme preparation has an inherent 3' leads to 5' exonuclease activity, attacking both native and denatured T7 DNA; it is free from detectable endonuclease activity.

Characterization of the membrane-bound inorganic pyrophosphatase in Rhodospirillum rubrum.

The membrane-bound inorganic pyrophosphatase (EC 3.6.1.1) from Rhodospirillum rubrum has been investigated with the tools of enzyme kinetics, and with two amino acid reagents, N-ethyl-maleimide (MalNET) and 4-chloro-7-nitrobenzofurazan (Nbf-Cl). 1. The concentration of the true substrate, MgPPi, was varied with constant concentrations of free Mg2+ or PPi. It was observed that Mg2+ acted as an activator. 2. Heat inactivation of the enzyme at 62 degrees C was slowed down in the presence of Mg2+. 3. MalNET and Nbf-Cl bind to the enzyme, and inhibit its activity. The effect of both reagents is dependent on the temperature. 4. A model is proposed where the 1:1 complex of Mg2+:PPi acts as substrate and Mg2+ interacts directly with the enzyme as an activator. PPi can bind to the enzyme, but is not hydrolyzed in the uncomplexed form.