Role of alkaline endonucleases in the release of soluble chromatin from thymus, spleen and liver nuclei of normal and irradiated mice.

Thymus, spleen and liver nuclei released a large fraction of soluble chromatin in vitro when incubation was carried out in sucrose media containing low concentrations of CaCl2 and/or MgCl2. A significant fraction of deoxyribopolynucleotides (DPN) was also extracted from nuclei. After 30 min of incubation at 37 degrees C, the maximum release of soluble chromatin was observed near a pH of 8, which corresponds to the optimum pH of the alkaline endonuclease activity from thymus, spleen and liver. The soluble chromatin and DPN were precipitated by increasing the bivalent ion concentration of the medium. The protein/DNA ratio and the molecular weight of DNA suggest that the soluble chromatin and DPN represent nucleosome-like particles. The release of soluble chromatin in the first 4 hours of incubation was significantly increased if the nuclear fraction was isolated from the thymus and spleen of whole-body irradiated mice (1000 rad). This effect was absent in the liver nuclei.     

Dynamics of the endo-DNAase activity of cell nuclei of mouse thymus and spleen lymphocytes during the immune response

Endo-DNAse (mostly Ca/Mg-dependent endonuclease) activity was studied in extracts of lymphocyte cellular nuclei from the spleen and thymus of mice upon their immunization with sheep red blood cells. Endo-DNAses were detected by their action on super-stranded DNA pBR 322. It has been established that endo-DNAse activity considerably changes in the course of immune response. The changes start in the early (induction) phase of immune response, are characterized by certain regularities and are distinct in thymus and spleen lymphocytes. It is assumed that endo-DNAses of lymphocyte cellular nuclei are involved in antigen-dependent lymphocyte differentiation.     

Mechanism of action of a mammalian DNA repair endonuclease

The mechanism of action of a DNA repair endonuclease isolated from calf thymus was determined. The calf thymus endonuclease possesses a substrate specificity nearly identical with that of Escherichia coli endonuclease III following DNA damage by high doses of UV light, osmium tetroxide, and other oxidizing agents. The calf thymus enzyme incises damaged DNA at sites of pyrimidines. A cytosine photoproduct was found to be the primary monobasic UV adduct. The calf thymus endonuclease and E. coli endonuclease III were found to possess similar, but not identical, DNA incision mechanisms. The mechanism of action of the calf thymus endonuclease was deduced by analysis of the 3' and 5' termini of the enzyme-generated DNA scission products with DNA sequencing methodologies and HPLC analysis of the material released by the enzyme following DNA damage. The calf thymus endonuclease removes UV light and osmium tetroxide damaged bases via an N-glycosylase activity followed by a 3' apurinic/apyrimidinic (AP) endonuclease activity. The calf thymus endonuclease also possesses a novel 5' AP endonuclease activity not possessed by endonuclease III. The product of this three-step mechanism is a nucleoside-free site flanked by 3'-and 5'-terminal phosphate groups. These results indicate the conservation of both substrate specificity and mechanism of action in the enzymatic removal of oxidative base damage between prokaryotes and eukaryotes. We propose the name redoxy endonucleases for this group of enzymes.     

Characterization of DNA kinase from calf thymus

DNA kinase has been purified to homogeneity from calf thymus. The purified enzyme, with a specific activity of 16.7 units/mg protein at 25 degrees C, exhibited a sharp pH/activity curve with a pH optimum at 5.5 and low activity at alkaline pH. The molecular weight of the enzyme was estimated by dodecylsulfate/polyacrylamide gel electrophoresis to be 5.4 X 10(4). The enzyme has a sedimentation coefficient of 4.0 S. An apparent molecular weight of 5.6 X 10(4) and a Stokes' radius of 3.3 nm were estimated by gel-filtration on Sephadex G-100. The enzyme phosphorylates neither yeast RNA nor poly(A) instead of DNA. Compared with rat liver DNA kinase, calf thymus DNA kinase is relatively resistant to the inhibition by sulfate (Ki = 7 mM) and pyrophosphate (Ki = 5 mM). The enzyme activity is markedly stimulated by polyamines at the sub-optimal concentration of Mg2+ but not by monovalent cations.     

Autodigestion of chromatin in some radiosensitive and radioresistant mouse cells. Role of proteolysis and endonucleolysis

Evidence is presented indicating that mouse thymus, spleen, kidney, lung and heart contain a protease activity with relatively high specificity for histones. It is suggested that degradation of chromatin occurring in irradiated lymphoid tissues is produced by the action of alkaline endonuclease in association with this histone protease. The autodigestion of chromatin was assessed by determining the release of soluble chromatin from cells suspended in sucrose media of low ionic strength. It was found that the protease inhibitors, phenylmethylsulphonyl fluoride and especially NaHSO3, were also capable of depressing the activity of alkaline endonuclease, the fragmentation of chromatin, and the release of soluble chromatin. The results suggest that the release of histones from irradiated lymphoid tissues cannot be considered as a determinant step in the fragmentation of DNA in chromatin.     

Evaluation of the mouse mutant "wasted" as an animal model for ataxia telangiectasia. I. Age-dependent and tissue-specific effects

The wasted mouse, an animal model proposed for the genetically transmitted human disease ataxia telangiectasia (AT), was examined for its biological, cytogenetic and biochemical properties. In affected homozygotes, a marked age-dependent decrease in the ratio of spleen and thymus to body weight, and a slight but significant decrease in the liver to body weight ratio were observed while no such change was found in the kidney. An age-dependent increase was observed in the frequency of both spontaneous and gamma-ray-induced chromosomal aberrations in bone marrow cells of wasted mice. In littermate control mice, neither of these alterations was observed in an age-dependent manner. The activity of a primer activating enzyme, which has been reported to be deficient in AT cells, also decreased with age in spleen cells, but not in liver cells of affected mice. However, alterations in apurinic DNA endonuclease activity were not detected in the developmental stages examined. These data indicate that this mouse mutant may serve as a useful animal model for studying the relationships between DNA repair and lymphoid tissue differentiation.     

BspLS2I--a new site-specific endonuclease from the thermophilic bacteria Bacillus species LS2]

A new restriction endonuclease BspLS2I was isolated from the thermophilic bacterium Bacillus species LS2 and purified by blue sepharose and hydroxyapatite chromatographies. The enzyme is an isoschizomer of SduI from Streptococcus durans. BspLS2I recognizes the sequence 5' G(G/A/T)GC(C/T/A) decreases C 3' on double-stranded DNA and cleaves it is indicated by the arrow to yield sticky-ended DNA fragments. Maximum catalytic activity of endonuclease was found in 10 mM tris-HCl (pH 7.9) in the presence of 15-30 mM MgCl2 at 50 degrees C. The phage T4 glucosylated DNA is not cleaved by the enzyme.     

Apurinic acid endonuclease activity from mouse epidermal cells.

An endonuclease activity making single-strand breaks into depurinated and alkylated DNA has been purified 500-fold from carcinogen-transformed mouse epidermal cells. The enzyme was active only at apurinic/apyrimidinic sites, regardless of whether they were produced by heating at an acidic pH or by alkylation with the ultimate carcinogen MeSO2OMe. The enzyme did not act on native DNA nor on ultraviolet-induced pyrimidine-dimers nor on steric distortions caused by modification of DNA with the carcinogen (Ac)2ONFln. The enzyme was active in the presence of 1 mM EDTA; however, at pH 7.4 optimal conditions were: 6mM MgCl2 and 40--120 mM KCl or 10--40 mM potassium phosphate. The enzyme eluted from hydroxyapatite, phosphocellulose and heparin-cellulose between 100--250 mM potassium phosphate but did not bind to DEAE-cellulose. Using four chromatographic steps the endonuclease was obtained free of exonuclease, demethylase and DNA glycosylase activity specific for DNA bases methylated with MeSO2OMe or MeNOUr. The molecular weight was 31 000 +/- 3000 as calculated from the diffusion coefficient (8.2 x 10-7 cm2/s) and the sedimentation value (2.7 S).     

Mouse spleen cell nuclear protein kinases and the stimulating effect of dsDNA on NHP phosphorylation by cyclic AMP-independent protein kinase in vitro

cAMP-dependent (designated as enzyme I, about 68,000 daltons) and cAMP-independent protein kinase (designated as enzyme II, about 45,000 daltons) have been partially purified from the nuclei of mouse spleen cells. Both kinases phosphorylated calf thymus histones as well as non-histone proteins (NHP) and required Mg2+ (8 mM) or Mn2+ (2 mM) for maximal activity. NEM (0.5 mM), which is an inhibitor of SH-enzymes, inhibited the histone phosphorylating activity of enzyme II by more than 90%, whereas it inhibited the activity of enzyme I by less than 10%. Moreover, the activity of enzyme II was more sensitive to high temperature than that of enzyme I. Non-histone protein (CM-III protein) served as a more effective substrate for enzyme II than histones; the Km value for CM-III protein was 34.4 micrograms/ml whereas that for histone H2a (14,300 daltons) was 155 micrograms/ml (1.08 x 10(-5) M). CM-III protein phosphorylation by enzyme II in vitro was greatly stimulated by the addition of dsDNA, but not by single-stranded DNA or bacterial ribosomal RNA. However, the phosphorylation of CM-III protein by enzyme I was less than 50% of that of histones, and there was no stimulatory effect. SDS-gel electrophoresis showed that two distinct NHPs (about 13,000 and 19,000 daltons) prepared from calf thymus chromatin were preferentially phosphorylated by enzyme II in vitro in the presence of dsDNA. This finding suggests that these two NHPs may be specific phosphate acceptors of cAMP-independent protein kinase (enzyme II) in the nuclei of mouse spleen cells.     

Isolation and analysis of Ca/Mg-dependent endonuclease from cell nuclei of human splenocytes

A scheme for the isolation of Ca,Mg-dependent endonuclease from human spleen lymphocyte nuclei has been developed. The isolation procedure resulted in protein preparations (Mr = 57 kD) possessing an enzymatic activity and stable upon storage for over a period of one year. The enzyme is an endonuclease which predominantly cleaves double-stranded DNA by a mixed single- and double-hit mechanism with the formation of 5'-phosphate and 3'-OH terminal groups. Its maximal activation is induced by Ca2+ plus Mg2+. The enzyme is also active in the presence of Mn2+, Ca2+, Mg2+ and Zn2+ and is inhibited by Co2+. NaCl and KCl (0.15-0.2 M) and p-chloromercuribenzoate (1 mM) also inhibit the enzyme. ATP has no activating effect.     

Ca2+, Mg2+-dependent endonuclease activity in different subpopulations of spleen cells from normal and erythroleukemic mice

We have detected Ca2+, Mg2+-dependent endonuclease activity in spleen cells of normal, Friend erythroleukemic, and phenylhydrazine-treated mice. When nuclei were isolated and incubated in the presence of Ca2+ and Mg2+ ions, the activity resulted in the production of 3'-OH termini in the cellular DNA and the release of chromatin due to internucleosomal DNA fragmentation. This enzyme activity was chromatin-bound and could be extracted from chromatin in an active form in 0.35 M KCl. The majority of endonuclease activity from erythroleukemic spleens was present in nuclei of precursor erythroid cells of low buoyant density (1.025-1.05 g/ml). Uninfected normal splenic tissue contained an endonuclease activity which was almost entirely confined to a B-lymphocyte population of high buoyant density (greater than 1.07 g/ml). Erythroid cell-enriched spleens from phenylhydrazine-treated mice exhibited a distribution of endonuclease activity in cells at low and high densities reflecting a mixture of erythroid and lymphoid cells. Cloned erythroleukemic cell lines propagated in vitro lacked cells of low density and showed no detectable endonuclease activity. However, nuclei from these cell lines were susceptible to exogenously added endonuclease extracted from erythroleukemic spleen cells. These same cell lines propagated as subcutaneous tumors contained endonuclease activity and a morphologically-similar low-density cell population which accounted for the endonuclease activity in these tumors. Nuclei from cloned lymphoid cell lines, representing different B-lymphocyte phenotypes, showed differences in the presence of endonuclease activity. Among the cell lines tested, only those expressing late B-cell markers showed detectable endonuclease activity.     

EcoRI activity: Enzyme modification or activation of accompanying endonuclease?

A study has been made of the factors and mechanism leading to appearance of the so-called EcoRI activity described by Polisky et al. (1975) in the restrictase EcoRI preparations. The preparations of purified restrictase EcoRI, precipitated at 0.9 ammonium sulphate saturation, as well as that obtained using standard techniques have been found to contain an admixture of an endonuclease which at neutral pH and high ionic strength multiply cleaves those DNAs which normally have only one recognition site for EcoRI. Under the standard conditions for EcoRI digestion this activity is found only when large amounts of freshly isolated enzyme are added to the incubation mixture and it is sharply enhanced by replacement of Mg2+ with Mn2+. The number and size of DNA fragments produced under such conditions practically do not differ from those found under the so-called EcoRI conditions, that is for alkaline pH values and low ionic strength. The optimum incubation mixture for the EcoRI activity has been found to be 10 mM Tris . HCl buffer (pH 8.8) + 2 mM Mn2+. Similar activity is induced also by addition to EcoRI solution of 40--50% glycerol or a number of organic solvents (dimethylacetamide (DMA), dimethylformamide (DMF), dimethylsulphoxide (DMSO), sulphalane (SP) in concentrations from 1 to 6%. The EcoRI activity induced by 50% glycerol or at alkaline pH values and low ionic strength is suppressed or sharply inhibited by 2--3 mM parachloromercuribenzoate (PCMB), while EcoRI is not sensitive to this agent. The DNA fragments cleaved by EcoRI have cohesive termini and can be easily ligated. It is suggested that the EcoRI activity can be due not only (or largely not) to modification of the "recognizing capacity" of the EcoRI restrictase but not activation of a latent specific endonuclease which is present in the restrictase preparation as an impurity.     

Tyrosine protein kinase activity of rat spleen and other tissues

Using a synthetic peptide (Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly) as a substrate, various normal tissues from the rat were probed for tyrosine protein kinase activity. Spleen was shown to contain much higher tyrosine protein kinase activity than other rat tissues (lung, brain, testes, liver, kidney, heart, and thymus, in decreasing order of specific activity). Most of the tyrosine protein kinase activity of the various rat tissues (greater than 80%) was present in the particulate fraction, and Nonidet P-40, a nonionic detergent, could activate the particulate form of the enzyme 2-20-fold in many of the tissues. Epidermal growth factor (1 microgram/ml), cyclic AMP, cyclic GMP, or Ca2+ did not increase spleen tyrosine protein kinase activity. Half-maximal enzyme activity was observed at 60-80 microM MgATP and at 2.2 mM peptide, and both Mg2+ (10 mM) and Mn2+ (0.5-1.0 mM) were effective divalent metal ions for the expression of activity. When the particulate fraction of spleen was incubated with [gamma-32P]ATP followed by polyacrylamide gel electrophoresis in the presence of Na dodecyl SO4, a number of alkali-stable bands were identified by autoradiography. Two major bands at Mr = 53,000 and 56,000 were shown to contain phosphotyrosine. Two similar alkali-stable bands containing phosphotyrosine but with lower amounts of 32P labeling were also observed in the particulate fractions of various other tissues (lung, brain, kidney, and testes). The particulate form of tyrosine protein kinase of rat spleen could be solubilized by using high concentrations of Nonidet P-40 (5%) at an alkaline pH (pH 9.0). Partial purification and subsequent filtration on Sephacryl S-200 yielded a peak of tyrosine protein kinase activity with an apparent molecular weight of 55,000. The two major phosphorylated bands of Mr = 53,000 and 56,000 co-migrated with the peak of enzyme activity. The solubilized and partially purified enzyme preparation phosphorylated only tyrosine residues when either endogenous proteins or casein were used as substrates. These results suggest that relatively high activities of tyrosine protein kinase exist in a normal tissue (rat spleen). Major endogenous substrates of the enzyme(s) appear to be represented by two proteins of Mr = 53,000 and 56,000; one or both of these substrates may be the tyrosine protein kinase itself.     

Apurinic endonuclease activity remains constant during early drosophila development

An endonuclease activity that acts on alkali-labile lesions in x-irradiated PM2 DNA and recognizes apurinic lesions in heat/acid treated DNA has been partially purified from Drosophila melanogaster embryos and its specific activity monitored throughout early development. The enzyme activity also showed a low level of activity on UV-irradiated DNA. The saturation kinetics observed with both x-irradiated and apurinic PM2 DNA substrates were similar. The endonuclease activity exhibited a broad pH optimum between pH 6 and 8.5 and was almost completely inhibited by 100 mM NaCl, 0.1 mM EDTA, 2 mM CaCl12 and 10 mM NEM. The reaction was not completely dependent on the presence of Mg⁺⁺cation, but optimum activity was obtained at a concentration of 0.1 mM; concentrations greater than 1 mM Mg^s++ were inhibitory. The specific activity of the apurinic endonuclease, partially purified from several stages of embryonic and early larval development, remained the same. Unfertilized eggs exhibited a reduced level of this presumptive repair activity.Abbreviations AP endonucleases Apurinic/apyrimidinic endonucleases     

Further purification and characterization of T4 endonuclease V.

T4 endonuclease V, which is involved in repair of ultraviolet-damaged DNA, has been purified 3600 fold from T4D-infected Escherichia coli. The enzyme shows optimal activity at pH 7.2 and does not require added divalent ions. Endonuclease V attacks both native and heat-denatured DNA provided that the DNA has been irradiated, and the enzyme activity is dependent on the dose of ultraviolet irradiation. The rate and the extent of the reaction are greater with irradiated native DNA although the Km values for the two types of DNA are the same (2.25 - 10(-5) M). The enzyme is readily inactivated by heat and is sensitive to p-chloromercuribenzoate. Endonuclease V-treated irradiated DNA is degraded by spleen phosphodiesterase only when the DNA has been treated with alkaline phosphatase, suggesting that the enzyme produces 5'-phosphoryl termini.     

Properties of an intracisternal A-particle-associated endonuclease activity which is stimulated by ATP.

An endonuclease activity associated with purified proteinase K-treated intracisternal A-particles was identified and characterized. The activity required divalent cations, preferring Mn2+ to Mg2+. Salt concentrations above 50 mM inhibited the activity. The endonuclease was greatly stimulated by ATP, ADP, and dATP, whereas AMP appeared to produce a slight inhibition. GTP had no apparent effect on the activity. The enzyme introduced single-stranded nicks into DNA and nicked preferentially supercoiled DNA duplexes in the presence of ATP, although linear duplexes also functioned as substrates. Single-stranded DNA was not nicked to any great extent. The molecular weight of the enzyme was estimated to be about 40,000. The characteristics of this enzyme are very similar to those of the endonuclease found associated with Friend murine leukemia virus.     

A mammalian nicking endonuclease.

Purification and properties are described for an endonuclease isolated from calf thymus which attacks double-stranded, unmodified DNA, primarily by making single-strand breaks. No detectable acid-soluble products arise from the reaction. Double-strand breaks may occasionally be produced by the introduction of single-strand breaks on opposite strands in close proximity. The enzyme does not attack denatured DNA and is not inhibited by tRNA. Although added divalent cations are not required for activity, the enzyme is inhibited by EDTA, which suggests an essential role for bound cations; reaction is inhibited by Ca2+. The endonuclease has a broad pH optimum and is inactivated by preincubation at temperatures of 45 degrees C and higher. The molecular weight as determined by gel chromatography is about 30 000. Analysis of the products of reaction on a defined substrate, bacteriophage T3 DNA, by sedimentation in alkaline sucrose density gradients indicates limit products with chain lengths of about 0.8 X 10(6) daltons. On electrophoresis in agarose gels these products were shown to be heterogeneous in size. The endonuclease appears to generate 3'-hydroxyl and 5'-phosphate ends. The ability of the endonuclease to utilize bovine DNA as substrate argues against a restriction role for this enzyme.     

Study of barley endonucleases and alpha-amylase genes

We have identified an endonuclease(s) that preferentially cleaves the internucleosomal linker regions in the aleurone chromatin producing mono- and oligonucleosomes. This enzyme(s) has been designated as a "linker"-specific nuclease(s). This nuclease does not require divalent cations for activity, and therefore it is not the "Ca2+-Mg2+-DNase" found in mammalian cells. The linker-specific nuclease activity is not detectable in the dry aleurone tissue and in the tissue treated with 0.5 mM cordycepin. The endonuclease activity of the aleurone tissue incubated with gibberellic acid is higher than the level of this endonuclease in tissue treated with abscisic acid or water alone. Nuclei isolated from embryos have lower levels of endonuclease activities compared to those from aleurone tissue. Digestion of the nuclei from embryos with micrococcal nuclease revealed the subunit structure of chromatin. In Southern blots of the HindIII digests of DNA from embryos, five DNA bands hybridized to a nick-translated alpha-amylase cDNA clone. In similar autoradiograms with aleurone DNA, particular bands are less visible, notably in the DNA isolated from the tissue treated with gibberellic acid. This is the first report of the presence of a linker-specific nuclease activity in plant cells.     

Purification and properties of a plant endonuclease specific for apurinic sites.

An endonuclease which hydrolyzes depurinated DNA has been isolated from Phaseolus multiflorus enbryos; it has a molecular weight around 40,000. The enzyme is specific for apurinic sites; it has no action on normal DNA strands or on alkylated sites, and is without exonulcease activity. The rate of phosphoester bond hydrolysis near apurinic sites is far greater in native than in denatured DNA. The endonuclease is not inactivated by 10 mM EDTA, but is activity is however stimulated by Mg2+ or Mn2+. Its optimum pH is 7.5 to 8.0, and its optimum temperature 40degrees although, at this temperature, it is rapidly denatured; even low NaCl concentrations inhibit the enzyme activity. The endonuclease for apurinic sites of P. multiflorus is a non-histone protein of chromatin; the properties (like thermosensitivity of susceptibility to ionic strength) of the enzyme in situ, working on chromatin DNA, might be different from those described for the isolated endonuclease in homogenous aqueous solution.     

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.