Isolation of Ca/Mg-dependent endonuclease of cell nuclei as the basic component in the chromatin nuclease complex of human lymphocytes

Highly purified preparations of Ca/Mg-dependent cell nuclear endonuclease have been obtained from human spleen lymphocytes. The purification was 2000-fold, and the enzyme was a polypeptide with a molecular weight of 57-54 kD. The study of its binding with monoclonal antibody enzyme, produced by various hybridoma strains obtained upon the immunization of mice with non-fractionated nuclear extract has shown that Ca/Mg-dependent endonuclease was the best expressed endonuclease of lymphocyte cellular nuclei. Possible production of some other cell nuclear endonucleases during processing of CH/Mg-dependent endonuclease is suggested.     

Specificity of fragmentation of DNA from pBR322 plasmid by Ca,Mg-dependent endonuclease from cell nuclei of human lymphocytes

Fragmentation of the plasmid pBR322 DNA by a purified preparation of Ca/Mg-dependent endonuclease has been studied. It was shown that on the first steps of reaction the double-stranded cuts are introduced into the superhelical DNA independent of singlestranded ones. The doublestranded cuts are introduced into superhelical and linear DNA in 12 sites enriched with GC-pairs, 9 of them include pentanucleotide CGCGG(CCGCC) that is functionally significant. Relaxation of the plasmid DNA by topoisomerase I blocks the sitespecific action of the enzyme. Ca/Mg-dependent endonuclease is concluded to be topologically dependent enzyme, possibly, participating in the recombination processes.     

The effect of irradiation and alkylating agents on chromatin breakdown in normal and malignant lymphoid cells

Regularities of chromatin degradation in thymocytes and LS/BL tumor cells have been investigated. It has been shown that the rate of DNA degradation by Ca/Mg-dependent endonuclease in LS/BL tumor cells is 25 times lower than that in thymocytes, and radiation does not induce chromatin degradation. The alkylating agent TS 160 causes chromatin degradation in both LS/BL cells and thymocytes. In contrast to radiation TS 160 inhibits the endogenous chromatin degradation by Ca/Mg-dependent endonuclease in thymocytes.     

Ca/Mg-dependent endonuclease as a probe for detecting chromatin changes in lymphocytes in chronic lymphoid leukemia

Chromatin fragmentation of bovine peripheral blood lymphocytes from normal animals and the ones suffering from chronic lympholeucosis (CLL) by DNase I, micrococcal nuclease and purified Ca/Mg-dependent endonuclease from nuclei of human splenocytes was studied. The lymphocytes chromatin from CLL animals was shown to be more resistant to nucleases, than the one from normal animals. It was found that difference between fragmentation of chromatin samples from normal and CLL bovines was more dramatic when Ca/Mg- dependent endonuclease was used versus traditionally exploited DNase I and micrococcal nuclease. The data suggest that purified Ca/Mg-dependent endonuclease can be a useful enzymatic probe for detection of lymphocytes chromatin changes during CLL.     

The nuclear desoxyribonuclease (DNAse) spectrum of normal rat thymus cells and after whole-body x-ray irradiation

Spectra of thymocyte nuclear DNAases of control and irradiated (4 Gy) rats have been investigated. Using the method of SDS-electrophoresis of nuclear proteins in DNA-polyacrylamide gel (PAAG) the authors managed to discover a number of polypeptides of 35, 32, 17.7, 17.2, and 16.4 kDA molecular mass possessing a DNAase activity. The enzyme of 35 kDA is only active in the presence of Ca2+ and Mg2+ ions. Nucleases of 32, 17.7, 17.2, and 16.4 kDA are active in the presence of Ca2+ ions and inactive in the presence of Mg2+ ions or in the absence of divalent cations. A simultaneous addition of Ca2+ and Mg2+ ions to the incubation medium causes a synergistic effect with respect to the manifestation of these DNAase activities. Nucleases of 32, 17.7, 17.2, and 16.4 kDa only emerge after the preliminary removal of histones by ion exchange chromatography on a column with CM-sephadex C-50. The enzymic activity of 32 kDA protein increases 60 min after irradiation and drops to the control value in 4 h. At the same time, the postirradiation increase in DNAase activity of a low-molecular weight enzyme group remains invariable throughout the entire period of observation (1-4 h). The preinjection of cycloheximide (CHI) prevents the postirradiation degradation of chromatin and, simultaneously, makes the enzymic activity, corresponding to 35 kDA protein, disappear at the electrophoregrams. The experiments with CHI permit to identify the given enzymic fraction as Ca/Mg-dependent endonuclease. This indicates the participation of normally pre-existing Ca/Mg-dependent endonuclease in implementing the process of chromatin enzymic degradation in the irradiated thymocytes.     

Changes in Ca, Mg-dependent endonuclease of DNA in isolated nuclei of human lymphocytes in lymphoproliferative diseases

Ca, Mg-dependent endonuclease is one of the main DNAses of lymphocyte chromatin. It's activity is known to increase in the immune response and to decrease in spontaneous and experimental CLL. These observations became a basis for analysis of possible clinical meaning of it's enzymatic activity assay. Donors' peripheral blood lymphocytes being tested, normal level of endonucleolysis for men and children was defined. Except that patients with different clinical forms of lymphoproliferative diseases such as chronic lympholeukemia, non-Hodgkin lymphomas, Hodgkin's disease were observed. The results showed that Ca, Mg-dependent endonucleolysis activity was decreased in comparison to donors' one. Ca, Mg-dependent endonucleolysis activity was the same in the group of patients with non-malignant pathology and in donors' one. Successful treatment and remission state of our patients was associated with alteration of the Ca, Mg-dependent endonucleolysis activity to normal level as well as immunological parameters. That is why the activity of Ca, Mg-dependent endonucleolysis is suggested to be a new criterion of immune state and lymphocyte malignant transformation.     

Use of monoclonal antibodies in comparative studies of Ca, Mg-dependent endonuclease in cell nuclei

Eight hybridoma cell lines derived from fusion between myeloma X-63 and mouse splenocytes were found to secrete monoclonal antibodies against Ca/Mg-dependent endonuclease of human spleen cell nuclei. Two of them, termed N and S, were used in comparative research of enzymes from different organs and species of animals. The data obtained show that N and S antibodies recognize different antigenic determinants of the enzyme molecule. Cross-reactions of antibodies with different antigens having similar antigenic determinants, exist in Ca/Mg-endonuclease of such species as man, mouse, rat and cattle. The evolutionary conservatism of this enzyme is suggested. The data show that the existence of tissue-specific (thymus-specific and spleen-specific) isoforms of Ca/Mg-endonuclease of cell nuclei is possible.     

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.     

Apurinic and apyrimidinic DNA endonuclease of extremely thermophilic Thermothrix thiopara.

An endonuclease specific for apurinic, apyrimidinic (AP) sites in DNA was purified nearly to homogeneity from the extremely thermophilic bacterium Thermothrix thiopara. The enzyme has a molecular weight of approximately 26,000. It cleaves neither native nor UV- or gamma-irradiated DNAs and has no contaminating exonuclease or uracil-DNA glycosylase activities. The enzyme has no cofactor requirement and is not inhibited by EDTA or N'-ethylmaleimide. It shows maximal activity at 70 degrees C and a pH between 7.5 and 9.0. The Arrhenius activation energy of the reaction is 17 kJ/mol, and the apparent Km for AP sites is 38 nM. The rate of heat inactivation of the enzyme followed first-order kinetics, with a half-life of 10 min at 70 degrees C but about 150 min in the presence of 0.5 M ammonium sulfate or 0.5 mg of bovine serum albumin per ml at the same temperature. One cell of T. thiopara contains sufficient AP endonuclease activity for hydrolysis of about 10(6) phosphodiester bonds per h at 70 degrees C. An extract of these bacteria does not contain detectable Mg-dependent AP endonuclease activity, and the above-mentioned enzyme appears to be the main AP endonuclease of T. thiopara.     

DNA recombination in vitro initiated by Ca/Mg-dependent endonuclease from cell nuclei of human splenocytes

The system of DNA recombination in vitro was constructed. It comprises two plasmids, the derivatives of pBR322 deleted in the genes for tetracycline resistance, and the recombinogenic extract of the thymus lymphocytes nuclei of mice. The system permits to study the effect of proteins and factors on the efficiency of recombination resulting in reconstruction of the tetracycline resistance gene. Double-strand cuts in one of the deleted plasmids were necessary for recombination. Double-strand cuts by Ca/Mg-dependent endonuclease of the human spleen lymphocytes nuclei were more efficient as compared with the ones of DNAase I, restriction endonucleases PaeI and SalI in the initiation of recombination. The possible role of Ca/Mg-dependent endonuclease in recombination in vivo is discussed.     

Internucleosomal chromatin degradation in myeloma and B-hybridoma cell cultures.

The activity of Ca/Mg-dependent endonuclease (CME) is strongly inhibited in myeloma X-63.Ag8.653 and B-hybridoma MLC-1c as compared with mouse splenocytes. Nevertheless, pronounced internucleosomal chromatin degradation occurs in both cell lines during long-term cultivation without passing. In isolated cell nuclei of X-63 the activation of CME, which precedes chromatin fragmentation in vivo and loss of cell viability, is revealed. The time-course of CME activation is opposite to cell proliferation and is not accompanied by alterations in enzyme quantity. The results suggest that cell death of X-63 and MLC-1c occurs via apoptosis, and involves the mechanisms controlling the activation and/or interaction of CME with chromatin.     

Role of a disorder in poly(ADP-ribosylation) in the activation of Ca/Mg-dependent endonuclease

The nuclei from the control and irradiated (3 h after irradiation at a dose of 10 Gy) thymocytes were preincubated with NAD in conditions optimal for poly (ADP) ribosylation. This was shown to decrease by 6-7- and 2-3 times, respectively, the rate of autolytic cleavage of DNA by Ca/Mg-dependent endonuclease. The inhibitors of poly (ADP-riboso)-polymerase, nicotine amide and thymidine, removed the effect of NAD. The data obtained prompt an assumption that the post-irradiation activation of Ca/Mg-nuclease in thymocytes is associated with the disturbance of its post-translation modification, poly(ADP)ribosylation.     

Analysis of class II (hydrolytic) and class I (beta-lyase) apurinic/apyrimidinic endonucleases with a synthetic DNA substrate.

We have developed simple and sensitive assays that distinguish the main classes of apurinic/apyrimidinic (AP) endonucleases: Class I enzymes that cleave on the 3' side of AP sites by beta-elimination, and Class II enzymes that cleave by hydrolysis on the 5' side. The distinction of the two types depends on the use of a synthetic DNA polymer that contains AP sites with 5'-[32P]phosphate residues. Using this approach, we now show directly that Escherichia coli endonuclease IV and human AP endonuclease are Class II enzymes, as inferred previously on the basis of indirect assays. The assay method does not exhibit significant interference by nonspecific nucleases or primary amines, which allows the ready determination of different AP endonuclease activities in crude cell extracts. In this way, we show that virtually all of the Class II AP endonuclease activity in E. coli can be accounted for by two enzymes: exonuclease III and endonuclease IV. In the yeast Saccharomyces cerevisiae, the Class II AP endonuclease activity is totally dependent on a single enzyme, the Apn1 protein, but there are probably multiple Class I enzymes. The versatility and ease of our approach should be useful for characterizing this important class of DNA repair enzymes in diverse systems.     

Resolution of the RNA editing gRNA-directed endonuclease from two other endonucleases of Trypanosoma brucei mitochondria.

RNA editing in kinetoplastids, the specific insertion and deletion of U residues, requires endonuclease cleavage of the pre-mRNA at each cycle of insertion/deletion. We have resolved three endoribonuclease activities from Trypanosoma brucei mitochondrial extracts that cleave CYb pre-mRNA specifically. One of these, which sediments at approximately 20S and is not affected substantially by DTT, has all the features of the editing endonuclease. It cleaves CYb pre-edited or partially edited mRNA only when annealed to the anchor region of a cognate guide RNA (gRNA), and it cleaves accurately just 5' of the duplex region. Its specificity is for the 5' end of extended duplex RNA regions, and this prevents cleavage of the gRNA or other positions in the mRNA. This gRNA-directed nuclease is evidently the same activity that functions in A6 pre-mRNA editing. However, it is distinct and separable from a previously observed DTT-requiring endonuclease that sediments similarly under certain conditions, but does not cleave precisely at the first editing site in either the presence or absence of a gRNA. The editing nuclease is also distinct from a DTT-inhibited endonuclease that cleaves numerous free pre-mRNAs at a common structure in the region of the first editing site.     

Effects of divalent metal ions on the activity and conformation of native and 3-fluorotyrosine-PvuII endonucleases.

The activities of restriction enzymes are important examples of Mg(II)-dependent hydrolysis of DNA. While a number of crystallographic studies of enzyme-DNA complexes have also involved metal ions, there have been no solution studies exploring the relationship between enzyme conformation and metal-ion binding in restriction enzymes. Using PvuII restriction endonuclease as a model system, we have successfully developed biosynthetic fluorination and NMR spectroscopy as a solution probe of restriction-enzyme conformation. The utility of this method is demonstrated with a study of metal-ion binding by PvuII endonuclease. Replacement of 74% (+/- 10%) of the Tyr residues in PvuII endonuclease by 3-fluorotyrosine produces an enzyme with Mg(II)-supported specific activity and sequence specificity that is indistinguishable from that of the native enzyme. Mn(II) supports residual activity of both the native and fluorinated enzymes; Ca(II) does not support activity in either enzyme, a result consistent with previous studies. 1H- and 19F-NMR spectroscopic studies reveal that while Mg(II) does not alter the enzyme conformation, the paramagnetic Mn(II) produces both short-range spectral broadening and longer range changes in chemical shift. Most interestingly, Ca(II) binding perturbs a larger number of different resonances than Mn(II). Coupled with earlier mutagenesis studies that place Ca(II) in the active site [Nastri, H. G., Evans, P.D., Walker, I.H. & Riggs, P.D. (1997) J. Biol. Chem. 272, 25761-25767], these data suggest that the enzyme makes conformational adjustments to accommodate the distinct geometric preferences of Ca(II) and may play a role in the inability of this metal ion to support activity in restriction enzymes.     

Characterization of a site-specific restriction endonuclease from Streptomyces aureofaciens.

A new type II sequence-specific restriction endonuclease, SauI, was isolated from Streptomyces aureofaciens IKA18/4. The purified enzyme was free of contaminating exonuclease and phosphatase activities. SauI cleaved lambda DNA at two sites, but did not cleave pBR322, simian virus 40, or phi X174 DNA. SauI recognized the septanucleotide sequence 5'-CCTNAGG-3' and cleaved at the position indicated by the arrow, producing a trinucleotide 5'-terminal extension.     

Gamma endonuclease of Micrococcus luteus: action on irradiated DNA

Gamma endonuclease is a Mg2+-independent enzyme of Micrococcus luteus that recognizes and cleaves DNA at a variety of altered pyrimidines produced by ionizing radiation. The production of enzyme-recognizable sites (ERS) by ionizing radiation under different irradiation conditions was measured. Ionizing radiation produced the greatest number of ERS when irradiations were performed under anoxic conditions in the presence of the free radical scavenger KI. Since dihydrothymine is a major pyrimidine lesion produced in DNA during anoxic irradiation, the ability of gamma endonuclease to excise this lesion was assessed. Dihydrothymine was released from DNA irradiated under anoxic conditions in a radiation dose-dependent manner, consistent with gamma endonuclease's known DNA glycosylase activity. Gamma endonuclease was also shown to cleave heavily uv-irradiated DNA. When the sequence specificity of gamma-endonuclease cleavage was studied using uv-irradiated DNA, cleavage was seen specifically at cytosines. The identity of this enzyme-recognizable cytosine photoproduct is not known.     

Action of Single-strand-specific S1 Endonuclease on Locally Altered Structures in Double-stranded DNA

The cleavage of double-stranded DNA by S1 endonuclease was studied by sucrose density gradient centrifugation analysis. The enzyme introduced no single-strand breaks into native T7 DNA under conditions where heat-denatured T7 DNA was completely degraded. By using enzyme at about 6 times higher the amount required for complete degradation of the heat-denatured DNA, it was possible to make a few single-strand breaks in native T7 DNA. Under the conditions where native T7 DNA is absolutely resistant to the enzyme, the susceptibility of locally altered structures naturally present and/or artificially induced in native double-stranded DNA to the enzyme was studied. It was evidenced that S1 endonuclease can cleave circular covalently closed, superhelical fl RFI DNA, depurinated T7 DNA, bleomycin-treated T7 DNA containing internal single-strand breaks, but not cleave intercalating drug-bound T7 DNA.     

Chromatin degradation in the death of thymic lymphocytes induced by radiation or dexamethasone: the necessity for a stage of preliminary proteolysis

Proteolytic activity in a protein fraction of a rat thymocyte nuclear matrix was found to increase 1-2 h after gamma-irradiation or administration of dexamethazone. Cycloheximide did not prevent the observed protease activation. Neither histons nor thymocyte nuclear matrix proteins were subjected to proteolysis after exposure to radiation or the hormone. Such proteolysis inhibitors as phenylmethylsulfonyl fluorine, trasilol, and partly leupeptine inhibited nuclear DNA degradation in irradiated and dexamethazone treated thymus lymphocytes. In all appearance, this effect was not due to Ca/Mg-dependent endonuclease inactivation. The same was observed in the system of autolytic chromatin degradation in isolated thymocyte nuclei.     

An endonuclease activity in human polymorphonuclear neutrophils that removes 8-hydroxyguanine residues from DNA+

An endonuclease that specifically removes 8-hydroxyguanine (oh8Gua) from DNA has been isolated from Escherichia coli. As the amount of oh8Gua produced in DNA of X-ray-irradiated mice is known to decrease with time after irradiation, an attempt was made to find a similar activity in human polymorphonuclear neutrophils (PMNs) using a synthetic dsDNA containing oh8Gua as a substrate. The PMN enzyme was isolated free of other DNases, and found to cleave the substrate DNA simultaneously at 2 sites, the phosphodiester bonds 5' and 3' to oh8Gua, producing free hydroxyl and phosphate groups, respectively. The enzyme showed almost no activity on DNAs containing other kinds of modified base tested or mismatched DNA. Thus human cells also contain an endonuclease that specifically removes oh8Gua residues from DNA.