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.     

Purification of an endonuclease from adenovirus-infected KB cells.

This report describes the purification of an endonuclease from extracts of adenovirus-type-2-infected KB cells. Endonuclease activity can also be detected in extracts of uninfected KB cells and the enzyme activities from extracts of uninfected and adenovirus-infected cells are very similar, if not identical. The enzyme has its maximal activity at pH 4.0. The enzyme found in uninfected and adenovirus-infectedcells is, however, strikingly different from an endonuclease isolated from calf serum. Hence, the endonuclease described is probably not a contaminant derived from the medium in which the KB cells were propagated. The endonuclease in crude extracts from uninfected or adenovirus-infected KB cells can be activated or its activity enhanced by treatment of the extracts with proteolytic enzymes, like pronase or trypsin. Evidence has been presented suggesting that this activation is due to proteolytic cleavage of an inhibitor present in crude extracts of uninfected and adenovirus-type-2-infected KB cells. A second endonuclease has been found in extracts of infected and uninfected cells with optimal activity at pH 7.2 and this endonuclease can be separated from the one with a pH optimum at 4.0.     

Purification and properties of a Bacillus subtilis endonuclease specific for apurinic sites in DNA.

An endonuclease which hydrolyzes depurinated DNA has been purified from extracts of Bacillus subtilis cells. The endonuclease is a monomeric protein and has a molecular weight of around 56,000. The enzyme is specific for apurinic sites in double-stranded DNA, has a pH optimum at 8.0, and is slightly stimulated with 50 mM NaCl but completely inhibited with 500 mM NaCl. It requires no divalent cations and is insensitive to EDTA; it has no associated exonuclease. These properties are very similar to those of Escherichia coli endonuclease IV, which is also insensitive to EDTA and has no exonuclease activity, and very different from those of the main endonuclease for apurinic sites (endonuclease IV) of the same bacterium.     

Purification and characterization of an apurinic/apyrimidinic endonuclease from HeLa cells

An endodeoxyribonuclease from HeLa cells acting on apurinic/apyrimidinic (AP) sites has been purified to apparent homogeneity as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The presence of Triton X-100 was necessary throughout the purification for stabilization and stimulation of activity. The endonuclease has an apparent native molecular weight of 32,000 determined by molecular sieving and an apparent subunit molecular weight of 41,000 as judged by its electrophoretic mobility in SDS-polyacrylamide gels. The activity has an absolute requirement for Mg2+ or Mn2+ and a broad pH optimum between 6.7 and 9.0 with maximal activity near pH 7.5. The enzyme has no detectable exonuclease activity, nor any endonuclease activity on untreated duplex or single-stranded DNA. It is inhibited by adenine, hypoxanthine, adenosine, AMP, ADP-ribose, and NAD+, but it is unaffected by caffeine, the pyrimidine bases, ADP, ATP, or NADH. The use of a variety of damaged DNA substrates provided no indication that the enzyme acts on other than AP sites. The enzyme appears to cleave AP DNA so as to leave deoxyribose-5-phosphate at the 5' terminus and a 3'-OH at the 3' terminus; it also removes deoxyribose-5-phosphate from AP DNA which has deoxyribose at the 3' terminus. Specific antibody has been produced in rabbits which interacts only with a 41,000-dalton protein present in the purified enzyme (presumably the enzyme itself), as well as with partially purified AP endonuclease fractions from human placenta and fibroblasts.     

Characterization of an endonuclease associated with the drug resistance plasmid pKM101.

An endonuclease was detected in strains of Salmonella typhimurium containing the drug resistance plasmid pKM101. The enzyme was not detectable in strains lacking this plasmid, but it was present in strains containing mutants of pKM101 that were no longer able to enhance host cell mutagenesis. The endonuclease had a molecular weight of roughly 75,000 and, at pH 7.0, was equally active on single-stranded and duplex deoxyribonucleic acid (DNA). The reaction with single-stranded DNA was optimal at pH 5.5, whereas with duplex DNA the optimum was pH 6.8. The enzyme required a divalent cation for activity, and it had no detectable exonuclease activity with single-stranded or duplex DNA. The endonuclease extensively degraded DNA with no apparent base specificity, forming 5'-phosphomonoester termini. Although characterization of the endonuclease has not revealed its function, the enzyme does not appear to be a restriction endonuclease.     

Characterization of a novel endonuclease from Crithidia fasciculata.

A new endonuclease activity has been identified in whole cell lysates of the trypanosomatid Crithidia fasciculata. This activity, termed endonuclease A (Endo A), introduces single-strand breaks at highly preferred sites in double stranded DNA substrates Physical analysis of this enzyme indicates that it has a sedimentation coefficient S20,W of 4.9 and a Stokes radius of 59A and thus, a native molecular weight of 125,000 and a frictional coefficient of 1.8. A monomeric structure is suggested for the enzyme based on the recovery of Endo A activity associated with a polypeptide with a molecular weight of 116,000-120,000, following electrophoresis on sodium dodecyl sulfate polyacrylamide gels. Endo A shows an absolute requirement for Mg2+ or Mn2+ and exhibits activity over a broad pH and temperature range, with optimal conditions for activity at pH 8.0 and 30 degrees C.     

NgoII, a restriction endonuclease from Neisseria gonorrhoeae.

EndoR . NgoII, a class II restriction endonuclease isolated from Neisseria gonorrhoeae, was purified to electrophoretic homogeneity. We were able to separate it from another restriction endonuclease of N. gonorrhoeae, NgoI, by phosphocellulose chromatography. NgoII is an isoschizomer of HaeIII, a restriction endonuclease of Haemophilus aegyptius, and was found to recognize the deoxyribonucleic acid nucleotide base sequence GGCC. NgoII was able to digest phage lambda deoxyribonucleic acid over a wide pH range, with optimal activity at pH 8.5. The enzyme has an absolute requirement for Mg2+; maximal enzyme activity was observed at 1 mM Mg2+. The active enzyme has a molecular weight of 65,000 and appears to be composed of six subunits of identical molecular weight (11,000). No methylase activity could be detected in the purified enzyme preparation.     

The negative charge of Glu-111 is required to activate the cleavage center of EcoRI endonuclease

King et al. (King, K., Benkovic, S. J., and Modrich, P. (1989) 264, 11807-11815) have shown that Glu-111 is required for DNA cleavage by EcoRI endonuclease and have suggested that this residue is required for activation of the cleavage center upon specific recognition. We have substituted Gln or Asp for Glu-111 by oligonucleotide-directed mutagenesis. First and second strand cleavage rate constants are reduced by a factor of more than 10(4) by the Gln-111 substitution. However, these rate constants are enhanced 9-fold when pH is increased from 7.6 to 8.5, which enhances strand cleavage at EcoRI sites by wild type endonuclease to a similar degree. The specific affinity of Gln-111 endonuclease for EcoRI sites is 1000 times greater than that of wild type enzyme reflecting a decrease in the rate constant governing specific complex dissociation. In contrast to Gln-111 endonuclease, the equilibrium specific affinity of Asp-111 endonuclease for the EcoRI sequence is similar to that of wild type enzyme, and first and second strand cleavage rate constants are reduced only 100-fold relative to wild type enzyme. These results suggest that a negative charge on residue 111 is required for strand cleavage and are consistent with participation of Glu-111 in activation of the DNA cleavage center, with energy associated with specific sequence recognition driving this process.     

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.     

Thymine glycol-DNA glycosylase/AP endonuclease of CEM-C1 lymphoblasts: a human analog of Escherichia coli endonuclease III

A thymine glycol-DNA glycosylase/AP endonuclease has been identified in human CEM-C1 lymphoblasts. The enzyme is active in the absence of divalent cations and has an apparent molecular size of approximately 60,000 daltons. The enzyme releases thymine glycol from osmium tetroxide-damaged DNA via an N-glycosylase activity and is associated with an endonuclease activity that mediates phosphodiester bond cleavage at sites of thymine glycol and apurinic sites. We propose that this enzyme, which we call redoxyendonuclease, is the human analog of a bacterial enzyme, E. coli endonuclease III, that recognizes oxidative DNA damage.     

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.     

A restriction endonuclease cleavage map of mouse mitochondrial DNA.

A restriction endonuclease cleavage map is presented for mouse mitochondrial DNA. This map was constructed by electron microscopic measurements on partial digests containing fixed D-loops, and by electrophoretic analysis of partial and complete single enzyme digests, and of double digests. No map differences were detected between mitochondrial DNA from cultured LA9 cells and an inbred mouse line for the six endonucleases used. Three cleavage sites recognized by HpaI, five sites recognized by HincII, two sites recognized PstI and four sites recognized by BamI were located with respect to the origin of replication and the EcoRI and HinIII sites previously determined by others. No cleavages were produced by KpnI or SalI. The migration of linear DNA with a molecular weight greater than 1 X 10(6) was not a linear function of log molecular weight in 1% agarose gels run at 6.6 volts/cm.     

A barley endonuclease specific for apurinic DNA. Isolation and partial characterization

An endonuclease specific for depurinated native DNA was isolated and partially purified from extracts of barley leaves. The procedure included streptomycin sulphate precipitation, ammonium sulphate fractionation, phosphocellulose, hydroxyapatite and Sephadex G-150 chromatography. Purity of the resulting enzyme was determined by gel electrophoresis and gel chromatography and specificity by testing the activity on intact and depurinated bacterial DNAs. At lower concentrations, the enzyme is specific for DNA containing apurinic sites. At higher concentrations, however, it degrades DNA in a non-specific manner. The nuclease has a pH optimum at 7.6, and a molecular weight of about 18000.     

Nuclease SP: a novel enzyme from spinach that incises damaged duplex DNA preferentially at sites of adenine.

A novel endonuclease has been isolated from extracts of spinach leaves (Spinacia oleracea). The enzyme has been purified by a series of column chromatography steps and has a molecular size of approximately 43,000 daltons. The spinach endonuclease cleaved double stranded DNA damaged by ultraviolet light or cis-diamminedichloroplatinum (II) primarily at sites of adenine when end-labelled DNA fragments of defined sequence were employed as substrates. The nature of the structural distortion contained in damaged, duplex DNA appears to be an important determinant for endonuclease cleavage. DNA helical distortions produced by UV light-induced (6-4) pyrimidine-pyrimidone photoproducts, but not cyclobutane pyrimidine dimers are recognized by the enzyme. The DNA cleavage products generated by the enzyme contain 3'-hydroxyl and 5'-phosphoryl termini. Single stranded DNA and RNA are hydrolyzed by the spinach endonuclease. This enzyme, which we call nuclease SP, is similar in several respects to other single-strand-specific nucleases such as N. crassa and mung bean nucleases and may function in DNA repair and/or recombination events in spinach cells. Nuclease SP should be a useful tool for the analysis of (6-4) photoproducts occurring in duplex DNA.     

Purification, properties and specificity of the restriction endonuclease from Bacillus stearothermophilus.

The restriction endonuclease BstI was purified from 70kg of Bacillus stearothermophilus. The final product is at least 97% pure as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; this major protein species co-migrates with the enzyme activity on native polyacrylamide-gel electrophoresis and isoelectric focusing. Pure restriction endonuclease BstI has a subunit mol.wt. of 26,000 and is probably a loosely associated dimer. The enzyme shows maximum activity at pH values between 7 and 9.5, and in the presence of 0.5-2mM-Mg2+. NaCl inhibits the restriction enzyme activity. Restriction endonuclease BstI cleaves DNA in a position identical with that cleaved by endonuclease BamHI (for Bacillus amyloliquefaciens), i.e.: (formula: see text). In the presence of high concentrations of enzyme, DNA cleavage occurs at secondary sites. This side-specificity is enhanced by the addition of glycerol. Preliminary studies indicate that these sites are of the type: (formula: see text).     

Partial purification and characterization of a human 3-methyladenine-DNA glycosylase.

A DNA glycosylase was purified about 30-fold from cultured human lymphoblasts (CCRF-CEM line) and was found to cleave 3-methyladenine from DNA alkylated with methyl methanesulfonate. The enzyme did not promote the release of 1-methyladenine, 7-methyladenine, or 7-methylguanine from DNA nor did it act on denatured methylated DNA. It produced apurinic sites in DNA alkylated with N-methyl-N-nitrosourea and ethyl methane-sulfonate as well as methyl methanesulfonate but not in untreated DNA or in DNA alkylated with nitrogen mustard or irradiated with ultraviolet light or X-rays. The glycosylase was free of detectable endonuclease activity in experiments with untreated DNA or DNA exposed to ultraviolet light; low levels of endonuclease activity, obtained when X-irradiated, alkylated, or depurinated DNA was the substrate, were attributed to contaminant apurinic endonuclease activity. This 3-methyladenine-DNA glycosylase has an estimated molecular weight of 34,000, is not dependent on divalent metal ions, and shows optimal activity at pH 7.5--8.5.     

Modulation of the DNA scanning activity of the Micrococcus luteus UV endonuclease

Micrococcus luteus UV endonuclease incises DNA at the sites of ultraviolet (UV) light-induced pyrimidine dimers. The mechanism of incision has been previously shown to be a glycosylic bond cleavage at the 5'-pyrimidine of the dimer followed by an apyrimidine endonuclease activity which cleaves the phosphodiester backbone between the pyrimidines. The process by which M. luteus UV endonuclease locates pyrimidine dimers within a population of UV-irradiated plasmids was shown to occur, in vitro, by a processive or "sliding" mechanism on non-target DNA as opposed to a distributive or "random hit" mechanism. Form I plasmid DNA containing 25 dimers per molecule was incubated with M. luteus UV endonuclease in time course reactions. The three topological forms of plasmid DNA generated were analyzed by agarose gel electrophoresis. When the enzyme encounters a pyrimidine dimer, it is significantly more likely to make only the glycosylase cleavage as opposed to making both the glycosylic and phosphodiester bond cleavages. Thus, plasmids are accumulated with many alkaline-labile sites relative to single-stranded breaks. In addition, reactions were performed at both pH 8.0 and pH 6.0, in the absence of NaCl, as well as 25,100, and 250 mM NaCl. The efficiency of the DNA scanning reaction was shown to be dependent on both the ionic strength and pH of the reaction. At low ionic strengths, the reaction was shown to proceed by a processive mechanism and shifted to a distributive mechanism as the ionic strength of the reaction increased. Processivity at pH 8.0 is shown to be more sensitive to increases in ionic strength than reactions performed at pH 6.0.     

Purification to homogeneity and characterization of a redoxyendonuclease from calf thymus.

A redoxyendonuclease from calf thymus was purified to apparent homogeneity. The redoxyendonuclease recognized and induced cleavage of DNA damaged by ultraviolet light. The enzyme preparation produced a single band of a relative molecular mass of approximately 34 kDa upon SDS/PAGE. The apurinic/apyrimidinic endonuclease and the DNA glycosylase activities remained associated in the apparently homogeneous preparation of the enzyme. The redoxyendonuclease activity displayed a broad pH optimum between pH 5.0-8.5 and exhibited no requirement for divalent cations. By application of FPLC columns Mono-S, Mono-Q and Mono-P, the isoelectric point (pI) of the enzyme was found to be approximately 8.0. Using the DNA sequencing procedure of Maxam and Gilbert [Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, 499-560] the purified enzyme was found to incise ultraviolet-light-irradiated DNA at pyrimidine sites as observed previously with a more crude form of the enzyme. While the most frequently cleavaged sites for the crude preparation were at cytosine residues, the apparently homogeneous enzyme preparation frequently induced cleavage sites at both cytosine and guanine residues. Predominant incision induced by the apparently homogeneous preparation was observed at guanine residues when a particular DNA sequence was used as substrate. Furthermore, the 16 N-terminal amino acid residues of the purified enzyme were identified. The sequence did not show any significant similarity to other known proteins.     

Glu-111 is required for activation of the DNA cleavage center of EcoRI endonuclease

Gap repair in the presence of 2'-deoxycytosine 5'-O-(1-thiotriphosphate) has been utilized to mutagenize the amino-terminal one-half of the structural gene for EcoRI endonuclease. This approach has led to identification of over 200 mutants defective in endonuclease function. One mutant protein, which binds to the EcoRI sequence but displays greatly reduced cleavage activity, is the consequence of a Glu to Gly change at position 111. This protein has been purified to homogeneity and characterized in detail. Subunit interactions governing the tetramer to dimer transition of the mutant endonuclease are near normal as are parameters governing its interaction with specific and nonspecific DNA sequences. However, the rate constants for first and second strand cleavage steps are reduced by 60,000- and 30,000-fold, respectively, as a consequence of the Glu----Gly change. The defect in chemical cleavage steps can be partially overcome by elevating the pH of the reaction buffer from 7.6 to 8.5, conditions which enhance the rate of EcoRI* strand cleavage by wild type enzyme to a similar degree. We suggest that the Glu-111 mutation affects an interface between recognition and cleavage functions of the enzyme, an idea consistent with the suggestion that the cleavage center of the endonuclease is subject to activation upon specific recognition of the EcoRI sequence.