Purification and properties of DNA endonucleases associated with Friend leukemia virus.

An endonuclease associated with the core of Friend leukemia virus (FLV) has been purified more than 10(3)-fold by ion exchange chromatography and gel filtration. Its molecular weight was determined by gel filtration to be about 40,000. Divalent cations were required for the endonuclease to function and KCl concentrations above 50 mM inhibited the enzyme activity. In the presence of Mg++ the purified enzyme nicked preferentially supercoiled circular DNA duplexes and in most of these molecules only one single-stranded nick was introduced per strand. The regions into which the nick could be introduced appeared to be randomly distributed on the circular molecule. When Mn++ was substituted for Mg++ the number of nicks introduced into DNA by the purified enzyme was greatly increased, and both relaxed circular and linear DNA duplexes were nicked as well as supercoiled circular DNA duplexes. Prior to its purification, however, in the presence of Mn++ the endonuclease activity in the virus extract was able to differentiate between circular and linear DNA duplexes, since both supercoiled and relaxed circular duplexes were nicked much more readily than linear duplexes. Single-stranded DNA functioned poorly as a substrate for the purified enzyme.     

Purification and characterization of DNA-relaxing enzyme from Haemophilus gallinarium.

A DNA-relaxing enzyme capable of concerted nicking and closing of DNA backbone bonds has been purified from Haemophilus gallinarum by two chromatographic steps and gel filtration. The enzyme efficiently catalyzes the removal of superhelical turns from a negatively twisted DNA and requires Mg2+ for this activity. Slight removal of superhelical turns from a positively twisted DNA generated by binding of ethidium bromide is found, but only at high enzyme concentrations. The DNA-relaxing activity is inhibited markedly with heat-denatured DNA, whereas native DNA and RNA have almost no affect on this activity.     

Properties of an endonuclease activity in Micrococcus luteus acting on gamma-irradiated DNA and on apurinic DNA

A protein fraction from Micrococcus luteus with endonuclease activity against gamma-irradiated DNA was isolated and characterized. An additional activity on apurinic sites could not be separated, either by sucrose gradient sedimentation or by gel filtration through Sephadex G 100. From gel filtration, a molecular weight of about 25 000 was calculated for both endonuclease activities. The endonuclease activity against gamma-irradiated DNA was stimulated five-fold with 5 mM Mg++, whereas that against apurinic sites was less dependent on the Mg++ concentration. 100 mM KCl inhibited the gamma-ray endonuclease, but not the apurinic endonuclease activity. In gamma-irradiated RNA the protein recognized 1.65 endonuclease sensitive sites per radiation induced single-strand break, among which are 0.45 alkali labile lesions in the nucleotide strand. The affinity of the enzyme for the endonuclease sensitive site was evaluated resulting in a Km-value of 73 nM.     

Pea chloroplast topoisomerase I: purification,characterization, and role in replication

A DNA-relaxing enzyme was purified 5 000-fold to homogeneity from isolated chloroplasts of Pisum sativum. The enzyme consists of a single polypeptide of 112 kDa. The enzyme was able to relax negatively supercoiled DNA in the absence of ATP. It is resistant to nalidixic acid and novobiocin, and causes a unit change in the linkage number of supercoiled DNA. The enzyme shows optimum activity at 37°C with 50 mM KCl and 10 mM MgCl₂. From these properties, the enzyme can be classified as a prokaryotic type I topoisomerase.Using a partiall purified pea chloroplast DNA polymerase fraction devoid of topoisomerase I activity for in vitro replication on clones containing the pea chloroplast DNA origins of replication, a 2–6-fold stimulation of replication activity was obtained when the purified topoisomerase I was added to the reaction at 70–100 mM KCl. However, when the same reaction was carried out at 125 mM KCl, which does not affect DNA polymerase activity on calf thymus DNA but is completely inhibitory for topoisomerase I activity, a 4-fold drop in activity resulted. Novobiocin, an inhibitor of topoisomerase II, was not found to inhibit the in vitro replication of chloroplast DNA.     

A DNA topoisomerase activity copurifies with the DNA polymerase induced by herpes simplex virus

A DNA-relaxing enzyme was found to copurify along with herpes simplex virus type I (HSV-1)-induced DNA polymerase throughout a multistep purification scheme. Both the enzymes had similar sedimentation velocity, required high ionic strength for optimal enzymatic activities and showed time dependence of reaction. The DNA-relaxing enzyme however, differed from the HSV-1 DNA polymerase in its requirement for higher Mg2+ concentration, rATP and much broader pH dependence. Furthermore, phosphonoacetic acid, a potent inhibitor of HSV-1 DNA polymerase did not influence the DNA-relaxing activity even at a much higher concentration. On the other hand, the DNA-relaxing enzyme associated with the DNA polymerase may be specified by HSV-1 since IgG fraction of rabbit antisera against the virus-infected cells but not against the mock-infected cells strongly inhibited both the enzymatic activities. Thus, HSV-1-induced DNA polymerase which is known to be associated with a 3' to 5' exonuclease may also be associated with yet another enzymatic activity involved in DNA metabolism.     

Tyrosyl-tRNA synthetase from wheat germ

Tyrosyl-tRNA synthetase (TyrRS) was purified 5,000-fold from wheat germ extract by ultracentrifugation, precipitation with ammonium acetate, and column chromatography. Under denaturing conditions the enzyme ran as a single band on SDS-polyacrylamide electrophoresis with an apparent Mr of 55,000. The native molecular weight determined by gel filtration was 110,000, suggesting a quaternary structure of an alpha 2 type for native TyrRS. Purified enzyme activity, based on the aminoacylation reaction, was studied in terms of Mg2+, ATP, pH, and KCl dependence. Optimum concentrations were 6 mM Mg2+, 4 mM ATP, and 200 mM KCl at pH 8. The Km values for ATP, tyrosine, and tRNA were 40, 3.3, and 1.5 microM, respectively. The instability of the TyrRS activity and the methods used for stabilizing it are discussed. In wheat germ extract we found a second tyrosylating activity that works with Escherichia coli tRNA, but not with wheat germ tRNA. We believe that this enzyme is the mitochondrial tyrosyl-tRNA synthetase of wheat germ.     

Purification and some properties of a deoxyribonucleic acid endonuclease endogenous to rat liver chromatin

A deoxyribonucleic acid (DNA) endonucleolytic activity has been purified from a 0.3 M KCl extract of rat liver chromatin by a combination of selective precipitation and ion-exchange and gel filtration chromatography. The purified protein has a molecular weight of 35 000 as determined by Sephadex G-200 gel filtration and sodium dodecyl sulfate-acrylamide gel electrophoresis. The nuclease activity is stimulated by the addition of Mg2+ and thus may represent the Mg2+-activated DNase endogenous to chromatin. The purified enzyme has the ability to make both single-strand nicks and double-strand cuts in DNA.     

DNA-dependent RNA polymerase from Pseudomonas aeruginosa

DNA-dependent RNA polymerase was purified from Pseudomonas aeruginosa. The subunit structure was typical of other eubacterial RNA polymerases in having beta' (157,000), beta (148,000), sigma (87,000), and alpha 2 (45,000) subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was dependent on Mg2+, displaying optimal activity at 10 mM MgCl2. Ca2+ and Zn2+ could not replace MgCl2 in the assay system, while Mn2+, produced partial activity. KCl at concentrations greater than 10 mM inhibited enzyme activity. Optimal enzyme activity was observed at pH 8.5-9.0. The RNA polymerase was stable in 50% (w/v) glycerol at 4 degrees C for more than 3 months. Enzyme activity was inhibited in vitro by heparin, streptolydigin, streptovaracin, actinomycin D, and rifampicin.     

Studies on the Processivity of Maize DNA Polymerase 2, an [alpha]-Type Enzyme

This paper describes studies on the processivity of an [alpha]-type DNA polymerase from maize (Zea mays L.) embryonic axes, designated as DNA polymerase 2. Using poly(dA)/oligo(dT) as template, DNA polymerase 2 has a processivity of 18 ([plus or minus]5) nucleotides incorporated, a value much lower than that found for wheat [alpha]-type DNA polymerase (P. Laquel, S. Litvak, M. Castroviejo [1993] Plant Physiol 102: 107-114). Conditions that maximally stimulate enzyme activity, such as 100 mM KCl and 12 mM Mg2+, are strongly inhibitory of processivity and cause the enzyme to become distributive under these conditions. Optimal concentrations for processivity are 10 mM KCl and 1 to 2 mM Mg2+. Both enzyme activity and processivity were found to be similar at different Mn2+ concentrations. Both DNA polymerase 2 activity and processivity are greatly reduced by spermine and N-ethylmaleimide. A distinguishing feature of processivity in DNA polymerase 2 was the response to ATP, which not only stimulated processivity by more than 2-fold, but also produced a distinctive pattern in which the enzyme seemed to pause every 10 nucleotides, reaching a value of 40 to 50 nucleotides incorporated. This pattern was observed in some, but not all, heparin-Sepharose fractions with enzyme activity, suggesting the possibility of different DNA polymerase 2 complexes.     

Laser flow measurements of scattering and fluorescence from cell nuclei in the presence of increasing Mg++ concentrations.

The effects of Mg++ on the spatial organization of nuclei from rat hepatocytes are analyzed in the range 0-60 mM, in the presence of suitable concentrations of KCl to reproduce physiological conditions. It is shown that the scatter-signal distribution measured by means of a flow microfluorimeter is greatly affected by this range of Mg concentrations. By coupling this result to phase-contrast-automated image analysis, it is possible to identify a shrinking process induced by Mg++ in the range 0-2.5 mM, which reaches a plateau in the range 5-20 mM and is followed by a swelling process in the range 30-60 mM. The same Mg ranges are shown to affect the intercalation of the fluorochrome acridine orange into chromatin, suggesting that the shrinking-swelling phenomenon has also a molecular correspondence at the genome level. Possible implications in terms of the influence of Mg++ on the organization of chromatin inside intact cells are briefly discussed.     

Purification and properties of a thermophilic and thermostable DNA polymerase from the archaebacterium Sulfolobus solfataricus

A DNA-dependent DNA polymerase was obtained in homogenous form from the thermoacidophilic archaebacterium Sulfolobus solfataricus. The enzyme, purified 706-fold, has a molecular mass of about 110000 daltons as determined by gel filtration and by glycerol gradient centrifugation. It requires Mg++ for its activity and has a pH optimum of 7.7. The activity is sharply dependent on the ionic strength. The enzyme is thermostable; its properties and activity requirements were characterized. The features of this enzyme are compared to those of other DNA polymerases isolated either from prokaryotes or eukaryotes.     

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.     

Isolation and characterization of BsuE methyltransferase, a CGCG specific DNA methyltransferase from Bacillus subtilis

The DNA methyltransferase M-BsuE that recognizes the sequence 5'-CGCG-3' has been isolated from Bacillus subtilis strain ISE15. A 1600-fold purification of M-BsuE was achieved by column chromatography on phosphocellulose, heparin-Sepharose, and DEAE-Sepharose. DNA methyltransferase activity was monitored in the column eluants radiochemically by the transfer of tritiated methyl groups from radiolabeled S-adenosylmethionine to poly(dGdC)-poly(dGdC) DNA, a sensitive and specific substrate for M-BsuE activity. The DNA sequence specificity of this methyltransferase activity was confirmed enzymatically by demonstrating that M-BsuE-methylated DNA was selectively protected from cleavage by the restriction enzyme isoschizomers, ThaI and FnuDII. Purified M-BsuE has an apparent molecular size of 41,000-43,000 as determined by gel filtration and migrates as a 41-kDa protein in a sodium dodecyl sulfate-polyacrylamide gel. DNA methylation by M-BsuE is dependent upon the presence of S-adenosylmethionine and 2-mercaptoethanol. M-BsuE methyltransferase activity is optimal at 37 degrees C in the presence of 50 mM Tris-HCl, pH 7.8, 25 mM KCl, 6 microM S-adenosylmethionine, 5 mM 2-mercaptoethanol, and 10 mM EDTA. M-BsuE methylates the external cytidine in its recognition sequence in both linear and supercoiled DNA. A unique property of M-BsuE is its ability to methylate 5'-CGCG-3' in Z-DNA.     

Purification and characterization of enolase from neuroblastoma cell line NG108

Neuroblastoma Cell line NG108 (a hybrid from Chinese hamster and mouse) produces high levels of enolase. Using ion-exchange chromatography and gel filtration, we have purified the enzyme (about 19 fold purification) and characterized it. The purified enzyme is a dimer of 90,000 m.wt. and is stable at room temperature. At higher temperatures (e.g., 50 degrees, 60 degrees C etc.) it gets inactivated. Enolase requires Mg++ for its activity and is resistant to urea. The optimum pH for the enzyme is 7, and Km values for Mg++ and 2-phosphoglycerate were found to be 3.1 and 1.1 mM, respectively. Fluorophosphate is a strong inhibitor of the enzyme. The clinical applications of the enzyme have been discussed.     

Isolation and properties of the SuaI restriction endonuclease from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius

A new restriction endonuclease SuaI was isolated from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. The enzyme is an isoschizomer of BspR1; it recognizes tetranucleotide GGCC and cleaves DNA in the center of this sequence. SuaI requires Mg2+, the optimal concentration being 6 mM. KCl at concentrations above 25 mM significantly inhibits the enzyme activity. The pH optimum lies within the range of 6--7 at 70 degrees C, the temperature optimum is at 70--75 degrees C. The enzyme is highly stable at temperatures up to 80 degrees C. DNA of S. acidocaldarius is not cleaved by the enzyme.     

Assembly of nucleosome-like structures mediated by cauliflower DNA topoisomerase

Nucleosome-like structures have been efficiently assembled in vitro by interaction of cauliflower histones, pBR322 DNA and cauliflower DNA topoisomerase, as assayed by supercoiling of relaxed circular DNA and by digestion with micrococcal nuclease. The optimum ionic strength for supercoiling was 150 mM KCl and the optimum weight ratio of histone to DNA was approximately 1.0. Four histones, H2A, H2B, H3 and H4, were necessary for the optimum assembling conditions, and the nucleosomes assembled protected DNA fragments of approximately 150 bp in length. It was found that cauliflower DNA topoisomerase acts not only as a DNA-relaxing enzyme but also as a chaperon factor for nucleosome assembly.     

A DNA topoisomerase activity copurifies with the DNA polymerase induced by herpes simplex virus

A DNA-relaxing enzyme was found to copurify along with herpes simplex virus type I (HSV-1)-induced DNA polymerase throughout a multistep purification scheme. Both the enzymes had similar sedimentation velocity, required high ionic strength for optimal enzymatic activities and showed time dependence of reaction. The DNA-relaxing enzyme however, differed from the HSV-1 DNA polymerase in its requirement for higher Mg²⁺ concentration, rATP and much broader pH dependence. Furthermore, phosphonoacetic acid, a potent inhibitor of HSV-1 DNA polymerase did not influence the DNA-relaxing activity even at a much higher concentration. On the other hand, the DNA-relaxing enzyme associated with the DNA polymerase may be specified by HSV-1 since IgG fraction of rabbit antisera against the virus-infected cells but not against the mock-infected cells strongly inhibited both the enzymatic activities. Thus, HSV-1-induced DNA polymerase which is known to be associated with a 3′ to 5′ exonuclease may also be associated with yet another enzymatic activity involved in DNA metabolism.     

Isolation and some properties of the site-specific endonuclease and methylase Bme2161 from Bacillus megaterium 216

The site-specific endonuclease Bme2161 was isolated as a homogeneous preparation by chromatography on phosphocellulose, hydroxyapatite and heparin-agarose. The molecular mass of the enzyme, determined by gel filtration and by electrophoresis under denaturing conditions, was found to be 60 kDa and 30 kDa respectively. These data indicate that the native enzyme consists of two identical subunits. The enzyme recognized the decreases pentanucleotide sequence 5'-GGACC-3' X 3'-CCTGG-5' and cleaves the sequence as indicated by arrows. The increases optimal concentration for endonuclease reaction is 6-7 mM Mg2+. The endonuclease relaxes its specificity in the presence of glycerol or dimethyl sulfoxide at low Mg2+ concentration (1-3 mM). Methylase Bme2161, which protects DNA against endonuclease Bme2161 action by DNA methylation, was isolated from the same bacterial strain.     

Characterization of a Mg2+-dependent phosphatase from turkey gizzard smooth muscle

A Mg2+-dependent phosphatase has been purified to apparent homogeneity from turkey gizzard smooth muscle. The enzyme has a Mr = 43,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 44,500 as determined by sedimentation equilibrium centrifugation under nondenaturing conditions. Using polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate all of the phosphatase activity was found to migrate as a single band, subsequently shown to have an Mr = 43,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is inactive in the absence of Mg2+ and maximum activity is reached at a free concentration of 12 mM Mg2+. Mn2+ can replace Mg2+, but the activity is only about one-fifth of that found with 12 mM Mg2+. NaF and the nucleotides ATP, ADP, and AMP inhibit phosphatase activity. This inhibition appears to be independent of their ability to bind Mg2+. The phosphatase purified from turkey smooth muscle appears to be identical with that purified from canine heart (Binstock, J. F., and Li, H. C. (1979) Biochem. Biophys. Res. Commun. 87, 1226-1234) and rat liver (Hiraga, A., Kikuchi, K., Tamura, S., and Tsuiki, S. (1981) Eur. J. Biochem. 119, 503-510).