Mn2+-dependent endonuclease activity of chromatin

The endogenous endonuclease activity of chromatin in isolated rat liver nuclei in the presence of Mn2+, Mg2+ and Ca2+ + Mg2+ was studied. The existence of a Mn2+-dependent endonuclease activity not coupled with the Ca2+, Mg2+-dependent endonuclease was demonstrated, which was weaker than the former one in isolated cell nuclei but higher than in the preparation of Ca2+, Mg2+-dependent nuclease obtained by gel filtration through Toyopearl HW 60F. The Mn2+-dependent splitting of chromatin predominantly occurs at linker DNA of distal parts of chromatin loops. A split-off of purified DNA was more universal than in the presence of Ca2+, Mg2+-dependent endonuclease; the hydrolysis rate of native and denaturated DNA appeared to be the same.     

Isolation of Ca2+, Mg2+-dependent nuclease from calf thymus chromatin

Ca2+,Mg2+-dependent nuclease was isolated from calf thymus chromatin by stepwise chromatography on DEAE-Sepharose, CM-Sephadex and DNA-Sepharose. The enzyme was purified more than 700-fold. SDS-PAGE electrophoresis revealed one protein band possessing an enzymatic activity. The molecular mass of the nuclease as determined by gel filtration is 25700 Da, that determined by 12% SDS polyacrylamide gel electrophoresis is 28,000 Da. In the presence of various ions the enzyme activity decreases in the following order: (Ca2+ + Mn2+) greater than (Ca2+ + Mg2+) greater than Mn2+; the pH optimum is at 8.0. In media with Mg2+, Ca2+, Co2+ and Zn2+ the nuclease is inactive. Some other properties of the enzyme are described.     

Purification and properties of an endodeoxyribonuclease from nuclei of bovine small intestinal mucosa

An endodeoxyribonuclease has been purified from nuclei of bovine small intestinal mucosa to a homogeneous state by a procedure involving affinity chromatography on heparin-agarose. The endonuclease, which was found to be bound to chromatin, has a pH optimum of 5.4. It requires Mn2+ or Co2+ for activity and its maximum activity with Mg2+ is about 80% of that with Mn2+. Its activity is strongly inhibited by sulfhydryl-blocking agents, and by ethidium bromide. The enzyme does not attack RNA and is inhibited by it. Its isoelectric point is 8.5 +/- 0.1, and its molecular weight is 49,000 +/- 3,000, determined by sucrose gradient sedimentation and gel filtration on Sephadex G-100. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the enzyme is composed of two nonidentical subunits with molecular weights of 30,000 and 23,000. The enzyme catalyzes the endonucleolytic cleavage of circular duplex ColE1 DNA via single strand scissions from the initial stage of degradation. The average size of the limit products of native phage T7 or ColE1 DNA is about 2,000 to 1,500 base pairs, estimated by neutral sucrose gradient sedimentation or agarose gel electrophoresis. The enzyme degrades denatured DNA about 20 times faster than native DNA. The products contain 5'-phosphoryl and 3'-hydroxyl termini, and all four deoxymononucleotides are present in almost equal amounts at the 5'-termini.     

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.     

Purification and characterization of an endonuclease from fruiting caps of basidiomycete Coprinus cinereus

An endonuclease was purified from the cap tissues of basidiocarp of Coprinus cinereus collected at early meiotic prophase. It has an optimal activity at pH 7.0 and 37 degrees C. It is a cationic enzyme with a molecular mass of 22 kDa by gel filtration, and contains a 12-kDa and a 14-kDa peptide as revealed by SDS gel electrophoresis and Western blot analysis. An antiserum was produced in rabbits against the purified Coprinus endonuclease. The specificity of this antiserum was demonstrated in a dot-blot analysis and, more critically, in an immunoinhibition of endonuclease activity. The Coprinus endonuclease requires Mg2+ and/or Ca2+ as co-factors. Ca2+ is more efficient than Mg2+ while the effect of combining both co-factors is the highest. The Coprinus endonuclease has a substrate preference for single-strand and supercoiled DNA. It gives only single-strand nicks on supercoiled DNA at low enzyme concentration and limited time of incubation. At high enzyme concentration and/or long incubation time, double-strand fragmentation occurred. As is discussed, this endonuclease is believed to be involved in the early phase of meiotic recombination.     

Purification and properties of deoxyribonuclease from human urine.

The DNAase in human urine was purified about 30-fold with a recovery of 28%. This involved DEAE-cellulose and phosphocellulose chromatography steps and gel filtration on Sephadex G-75. The enzyme required divalent cations such as Co2+, Mg2+, Mn2+ and Zn2+ for activity, but Ca2+, Cu2+ and Fe2+ were ineffective. EDTA and G-actin inhibited the reaction. The maximum activity was observed at pH 5.5 in acetate buffer plus Co2+ or Mg2+ and Ca2+. It had a molecular weight of approximately 38 000, estimated by gel filtration on Sephadex G-75 and isoelectric point of around pH 3.9. The enzyme is an endonuclease which hydrolyzes native, double-stranded DNA about 3 to 4 times faster than thermally denatured DNA to produce 5'-phosphoryl- and 3'-hydroxyl-terminated oligonucleotides. The final preparation was free of non-specific acid and alkaline phosphatases, phosphodiesterase and ribonuclease activities.     

Specificity of cyclic GMP activation of a multi-substrate cyclic nucleotide phosphodiesterase from rat liver

Phosphoprotein phosphatase IA, which represents the major glycogen synthase phosphatase activity in rat liver cytosol, has been purified to apparent homogeneity by chromatography on DEAE-cellulose, histone - Sepharose-4B and Sephadex G-100. The molecular weight of the purified enzyme was 40 000 by gel filtration and 48 000 by sodium dodecyl sulfate gel electrophoresis, Phosphatase IA is therefore a monomeric protein. When treated with 80% ethanol at room temperature, phosphatase IA underwent an inactivation which was totally prevented by 2 mM MgCl2. Catalytically, phosphatase IA has a preference for glycogen synthase D compared with phosphatases IB and II and obligatorily requires Mg2+ or Mn2+ for activity. Maximum activity was attained at 5 mM MgCl2. Since Mg2+ does not activate other phosphoprotein phosphatases in rat liver cytosol, we propose the term 'Mg2+-dependent glycogen synthase phosphatase' for phosphatase IA.     

Purification and characterization of the sesquiterpene cyclase trichodiene synthetase from Fusarium sporotrichioides

The sesquiterpene cyclase, trichodiene synthetase, has been purified from a supernatant fraction of Fusarium sporotrichioides by hydrophobic interaction, anion exchange, and gel filtration chromatography. Purified enzyme had a specific activity 15-fold higher than that previously reported for preparations of terpene cyclases. Molecular weight determinations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography indicated the enzyme to be a dimer with a subunit of Mr 45,000. The requirement of Mg2+ (Km 0.1 mM) for activity could be partially substituted with Mn2+ at a concentration of 0.01 mM, but higher concentrations of Mn2+ were inhibitory. Maximum activity was observed between pH 6.75 and pH 7.75. The Km for farnesyl pyrophosphate was 0.065 microM.     

Purification and characterization of DNA-dependent ATP phosphohydrolases from KB cells

DNA-dependent ATPases have been purified from logarithmically growing KB cells by chromatography on single-stranded DNA cellulose and phosphocellulose. Phosphocellulose resolved the DNA-dependent ATPases into three activities designated ATPase I, II and III, respectively. From gel filtration and sedimentation analysis ATPases II and III were found to be very similar, both with calculated molecular weights of 78,000. Due to the extreme lability these enzymes were not purified further. The molecular weight of ATPase I determined by gel filtration and sedimentation analysis was calculated to be 140,000. ATPase I was further purified by gradient elution on ATP-agarose, revealing two peaks of activity (IA and IB), and by sucrose gradient sedimentation. Analysis of the fractions from the sucrose gradient by sodium dodecylsulphate gel electrophoresis revealed only one broad polypeptide band co-sedimenting with both ATPase IA and ATPase IB. This band was composed of four closely spaced polypeptides with apparent molecular weights of 66,000, 68,000, 70,000 and 71,000. Comparison of the native molecule weight (140,000) with these results suggests that ATPase I is a dimer. ATPase IA and IB were indistinguishable in their structural and enzymatic properties and presumably represent the same enzyme. The purified enzyme has an apparent Km of 0.5 mM for ATP producing ADP + Pi. A maximum activity of 2,100 molecules of ATP hydrolyzed per enzyme molecular per minute was found. Hydrolysis of ATP requires the presence of divalent cations (Mg2+ greater than Ca2+ greater than Mn2+ greater than Co2+). A broad pH optimum (pH 6--8) was observed. The enzyme uses ATP or dATP preferentially as a substrate, while other deoxyribonucleoside or ribonucleoside triphosphates were inactive. ATPase I prefers denatured DNA as cofactor. The activity with native DNA is 40% of that with denatured DNA.     

Cell nuclear DNases: multiplicity and heterogeneity

In order to determine the ratio of activities of major endonucleases of rat liver chromatin, a stepwise fractionation of cell nuclear extracts by chromatography on phosphocellulose and gel filtration through Toyopearl HW60 was carried out. This procedure resulted in partially purified preparations of Ca2+,Mg2+-dependent endonuclease (55 +/- 10 kD), Ca2+,Mg2+-dependent endonuclease (30 +/- 10 kD), Mn2+-dependent endonuclease (30 +/- 5 kD) and acid cation-independent endonuclease. The Ca2+,Mg2+-dependent endonuclease with Mr of 55 +/- 10 kD made up to 57% of the nuclear extract activity in the presence of Ca2+ + Mg2+ and revealed a high calcium-magnesium synergism. Under the same experimental conditions, the 30 +/- 10 kD enzyme made up to 33% of the nuclear extract activity and revealed a low synergism. The activity of Mn2+-dependent endonuclease made up to 26% of the total nuclear extract activity in the presence of Mn2+, that of acid endonuclease--11% of the extract activity in 1 mM EDTA at pH 5.0. It was assumed that the low molecular weight Ca2+,Mg2+-dependent endonuclease represents a product of limited proteolysis of high molecular weight Ca2+,Mg2+-dependent endonuclease.     

Purification and properties of the major apurinic/apyrimidinic endodeoxyribonuclease of rat-liver chromatin

Two nucleases active on alkylated-depurinated DNA have been extracted from rat liver chromatin with 1 M KCl. The major enzyme was purified to near homogeneity; it has a molecular weight of 12 500 (although some dimerization might occur), needs Mg2+ or Mn2+ for activity. The endonuclease activity is specific for apurinic/apyrimidinic sites in DNA; the enzyme has no associated exonuclease activity.     

Purification and properties of magnesium- and manganese-dependent ribonucleases H from chick embryo

Two RNases H, Mg2+- and Mn2+-dependent RNases H, are present in extracts of chick embryo. These RNases H can be separated by phosphocellulose column chromatography. Mg2+-dependent RNase H was purified over 900-fold and Mn2+-dependent RNase H over 1,700-fold from chick embryo extracts. The molecular weight of the purified Mg2+-dependent RNase H was about 40,000 and of the Mn2+-dependent RNase H about 120,000, when estimated by gel filtration. Mg2+-dependent RNase H exhibits maximal activity at pH 9.5, and requires 15 to 20 mM Mg2+ for maximal activity, whereas Mn2+-dependent RNase H is most active at pH 8.5, and is maximally active at the concentration of 0.4 mM Mn2+, and has some activity with Mg2+. Both enzymes require a sulfhydryl reagent for maximal activity. Mn2+-dependent RNase H was inhibited by o-phenanthroline, pyrophosphate, and those polyamines tested, whereas Mg2+-dependent enzyme was not, although it was inhibited by NaF. Both RNases H liberate a mixture of oligonucleotides with 5'-phosphate and 3'-hydroxyl termini endonucleolytically.     

Purificantion and characterization of inorganic pyrophosphatase from Thiobacillus thiooxidans.

An inorganic pyrophosphatase [EC 3.6.1.1] was isolated from Thiobacillus thiooxidans and purified 975-fold to a state of apparent homogeneity. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate and no activity was found with a variety of other phosphate esters. The cation Mg2+ was required for maximum activity; Co2+ and Mn2+ supported 25 per cent and 10.6 per cent of the activity with Mg2+, respectively. The pH optimum was 8.8. The molecular weight was estimated to be 88,000 by gel filtration and SDS gel electrophoresis, and the enzyme consisted of four identical subunits. The isoelectric point was found to be 5.05. The enzyme was exceptionally heat-stable in the presence of 0.01 M Mg2+.     

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 of a eukaryotic site-specific endonuclease, Endo.Sce I, from Saccharomyces cerevisiae and effectors on its specificity and activity

A site-specific endonuclease (Endo.Sce I) which caused double-strand scission of DNA was highly purified from a eukaryote, Saccharomyces cerevisiae IAM4274. The molecular weight of the active form of Endo.Sce I was estimated to be 120,000 and 110,000 by sedimentation analysis on a glycerol density gradient and gel filtration on Ultrogel AcA34, respectively. Analysis of the fractions from the last column chromatography by polyacrylamide gel-electrophoresis in the presence of sodium dodecyl sulfate and by an assay of the endonucleolytic activities suggested that Endo.Sce I consists of two non-identical subunits with molecular weights of 75,000 and 50,000. Unlike restriction endonucleases, Endo.Sce I was active on chromosomal DNA of the cells which produced Endo.Sce I. Single-stranded DNA was not cleaved by Endo.Sce I, but inhibited the endonucleolytic activity of the enzyme on double-stranded DNA. The endonucleolytic activity of Endo.Sce I required the magnesium ions (Mg2+) as a sole cofactor; Mg2+ could not be replaced by Ca2+ or Zn2+. When Mg2+ was replaced by manganese ions (Mn2+), extensively purified Endo.Sce I cleaved double-stranded DNA at many other sites in addition to the sites at which DNA was cleaved in the presence of Mg2+. Experiments indicated that this is not the activation of contaminating endonuclease in the preparation of Endo.Sce I, but the result of relaxation in the site-specificity of cleavage.     

Partial purification and characterization of type I DNA topoisomerase from Bacillus stearothermophilus

Type I DNA topoisomerase was partially purified from Bacillus stearothermophilus by ammonium sulfate precipitation and column chromatographies on phosphocellulose, DEAE-cellulose and heparin-agarose. On heparin-agarose chromatography, topoisomerase I activity was separated into three fractions (designated Fractions A, B, and C). Each fraction was further subjected to gel filtration on Sephacryl S-200. From electrophoretic analysis on polyacrylamide gel, Fraction A was found to contain two enzyme species having molecular weights of 110,000 and 100,000, and Fraction B one enzyme species with a molecular weight of 80,000. The molecular weight of the enzyme in Fraction C was estimated to be around 150,000 by gel filtration. The enzymes in Fractions A and B exhibited little activity in the presence of Mg2+, while the activity was increased remarkably by NaCl with Mg2+. No activity was observed in the presence of NaCl alone. The enzyme in Fraction C required only Mg2+ for full activity. With Fraction A, the topoisomerase I-induced cleavage sites on tetracycline-resistant plasmid pNS1 (2.55 megadaltons) were mapped. Fraction A cleaved the DNA at ten specific sites. These sites were compared to those of the Haemophilus gallinarum enzyme, which have already been mapped (Shishido et al. (1983) Biochem. Biophys. Acta 740, 108). The results showed that there is a remarkably coincidence between the cleavage sites induced by the B. stearothermophilus and H. gallinarum enzymes.     

Purification and amino-terminal amino acid sequence of an apurinic/apyrimidinic endonuclease from calf thymus.

An apurinic/apyrimidinic (AP) endonuclease (E.C.3.1.25.2) has been purified 1100 fold to apparent homogeneity from calf thymus by a series of ion exchange, gel filtration and hydrophobic interaction chromatographies. The purified AP endonuclease is a monomeric protein with an apparent molecular weight on SDS-PAGE of 37,000. On gel filtration the protein behaves as a protein of apparent molecular weight 40,000. DNA cleavage by this AP endonuclease is dependent on the presence of AP sites in the DNA. DNA cleavage requires the divalent cation Mg2+ and has a broad pH optimum of 7.5-9.0. Maximal rates of catalysis occur at NaCl or KCl concentrations of 25-50 mM. The amino acid composition and the amino-terminal amino acid sequence for this AP endonuclease are presented. Comparison of the properties of this AP endonuclease purified from calf thymus with the reported properties of the human AP endonuclease purified from HeLa cells or placenta indicate that the properties of such an AP endonuclease are highly conserved in these two mammalian species.     

Mg2+-dependent/poly(ADP-ribose)-sensitive endonuclease

A novel endonuclease from adult hen liver nuclei has been purified to a homogeneous state through salt extraction, ammonium sulfate fractionation, gel filtration, acetone fractionation, and successive chromatography of 1) hydroxyapatite and DNA Sepharose and 2) hydroxyapatite and isoelectric focusing. The endonuclease has a pH optimum at 9.0 and requires Mg2+ for activity. The enzyme hydrolyzes more rapidly in the order of polynucleotide: denatured DNA = rRNA greater than poly(dA) = poly(dT) greater than poly(dC) = poly(dG) greater than native DNA. This endonuclease degrades denatured DNA about 20 times more rapidly than does the native DNA. The products contain 5'-phosphoryl and 3'-hydroxyl termini and all four deoxynucleotides are present while dGMP is predominant. The enzyme cleaves the circular duplex PM2 DNA, endonucleotically, via single strand scission. The isoelectric point is 10.2 +/- 0.2 and the molecular weight is 43,000 +/- 2,000, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. Pyridoxal 5'-phosphate and 2,3-butanedione inhibit the catalytic activity, respectively. The inhibition of DNA binding activity was also seen with former, but not with the latter. Purified Mg2+-dependent alkaline endonuclease was used to investigate the nature of poly(ADP-ribose) inhibition of the enzyme. In contrast to the Ca2+/Mg2+-dependent endonuclease (Yoshihara, K., Tanigawa, Y., Burzio, L., and Koide, S. S. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 289-293), ADP-ribosylation of the endonuclease protein was not observed. When 100 ng of the poly(ADP-ribose) having four to five ADP-ribose units per molecule were added to the nuclease assay system (total volume of 0.2 ml) 14% inhibition was observed, and increase in the chain length increased the inhibition. When 100 ng of poly(ADP-ribose) consisting of 20 or more units of the ADP-ribose per mol were added, the inhibition was over 95%. The possible role of the poly(ADP-ribose)-sensitive endonuclease is discussed.     

Partial purification and characterization of CTP:cholinephosphate cytidylyltransferase from castor bean endosperm

CTP: cholinephosphate cytidylyltransferase (EC 2.7.7.15) has been purified approximately 600-fold from postgermination endosperm of castor bean. The enzyme was solubilized with n-octyl beta-D-glucopyranoside and then subjected to ion exchange and gel filtration chromatography. The Km's of the purified enzymatic activity were 0.37 and 1.1 mM for CTP and choline phosphate, respectively. Magnesium was required for activity. The purified cytidylyltransferase activity was inhibited by both phosphate and ATP. The extent of ATP inhibition was dependent on preincubation time, temperature, and Mg2+ and Ca2+ concentrations. The possible regulation of cytidylyltransferase in castor bean endosperm by protein phosphorylation is discussed.     

A cofactor protein required for actin activation of myosin Mg2+ATPase activity in leukemic myeloblasts

The Mg2+ATPase activity of the myosin of a myeloid leukemia cell line (Ml) was not activated by purified Ml actin or by muscle actin alone. Activation required the presence of a cellular fraction as a cofactor in addition to the actin, when Mg2+ATPase was stimulated as much as 20-fold. The cofactor was partially purified and characterized. 1) Its molecular weight was estimated as 45,000 to 55,000 daltons by gel filtration and as 45,000 daltons by SDS polyacrylamide gel electrophoresis. 2) The cofactor was a light chain kinase that phosphorylated both the L1 and L2 light chains of the Ml cell myosin, but not the L3 or heavy chain.