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.     

Purification and properties of a nuclear DNA endonuclease from HeLa S3 cells.

An endonuclease that can act on calf thymus DNA and circular doublestranded phage PM2 DNA has been isolated from HeLa S3 cell chromatin. Approximately 200-fold purification was achieved by a sequence of subcellular fractionation, differential NaCl solubility and chromatography on CM-Sephadex, DEAE-cellulose and hydroxyapatite, and isoelectric point is pH 5.1 +/- 0.2. Divalent cations are necessary for its activity and the enzyme is heat inactivated at 60 degrees C. The enzyme activity is sensitive to caffeine and sulfhydryl reacting compounds. The molecular weight, determined by gel filtration and SDS gel electrophoresis, is approx. 22 000.     

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.     

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.     

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.     

Endoplasmic reticulum nuclease. Purification and specificity

An endonuclease, which was originally identified for its RNA polymerase inhibitory activity, was isolated from rat liver endoplasmic reticulum. The enzyme yields on gel chromatography four active fractions of different molecular weights (Mr 5.3 X 10(4), 9 X 10(4), 1.55 X 10(5) and Sephacryl S-200 fraction at V0). Each fraction contains polypeptide chains which give a single band on sodium dodecylsulphate electrophoresis (Mr 5.4 X 10(4). This indicates that the enzyme is an oligomeric protein and each of its subunits exhibits the same or very similar molecular weights. Deoxyribonucleoside and ribonucleoside triphosphates can bind to the endoplasmic reticulum nuclease. Binding is enhanced in the presence of divalent cations particularly Mg2+. The enzyme exhibits mainly RNase activity but can also degrade denatured DNA and DNA . RNA hybrids which contain breaks in one of the two strands. Poly(A) and mainly poly(U) are most susceptible to its nucleolytic activity whereas poly(C) is completely resistant.     

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 and properties of DNA endonucleases associated with Friend leukemia virus.

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

Purification and characterization of a mitochondrial endonuclease from Drosophila melanogaster embryos.

A mitochondrial endonuclease from Drosophila melanogaster embryos was purified to near homogeneity by successive fractionation with DEAE-cellulose and heparin--avidgel-F, followed by FPLC chromatography on mono S, Superose 12 and a second mono S column. This enzyme digests double-stranded DNA more efficiently than heat-denatured DNA. The endonuclease activity has a molecular mass of 44 kDa, as determined under native conditions using a gel-filtration Superose 12 column. The prominent peptide detected by SDS/polyacrylamide gel electrophoresis likewise has a molecular mass of 44 kDa, suggesting a monomeric protein. The enzyme has an absolute requirement for divalent cations, preferring Mg2+ over Mn2+. No activity could be detected when these cations were replaced by Ca2+ or Zn2+. The pH optimum for this enzyme activity is 6.5-7.4 and its isoelectric point is 4.9. Both single-strand and double-strand breaks are introduced simultaneously into a supercoiled substrate in the presence of MgCl2 or MnCl2. Endonuclease-treated DNA serves as a substrate for DNA polymerase I from Escherichia coli, suggesting that 3'-OH termini are generated during cleavage. The enzyme is free from any detectable DNA exonuclease activity but not from RNase activity. Partial inhibition by antibodies raised against mitochondrial endonucleases derived from bovine heart and Saccharomyces cerevisiae have revealed a potential structural homology between these nucleases.     

Bleomycin-treated DNA is specifically cleaved only by endonuclease IV in E. coli.

We have isolated an endonuclease from E. coli active on bleomycin-treated DNA. Purification on DEAE-cellulose separated this activity in strains lacking endonuclease I, endonuclease III or exonuclease III. After DEAE chromatography, the enzyme was active in the absence of divalent cations and was not inhibited by tRNA or harmane. In addition, this enzyme was stable at 45 degrees C for 20 min. These properties are consistent with this activity being endonuclease IV. This was supported by our finding no activity in a strain lacking endonuclease IV.     

Ribonuclease H from rat liver. I. Partial purification and characterization of nuclear ribonuclease H1.

A ribonuclease H, an enzyme that specifically degrades the RNA moiety of RNA-DNA hybrid, has been partially purified from rat liver nuclei and characterized. Neither native or denatured DNA, nor single or double-stranded synthetic polyribonucleotides were degraded by the enzyme. The enzyme possesses a molecular weight of about 36,000 and requires alkaline pH, magnesium ions, and ammonium sulphate for maximum activity. The enzyme acts on the hybrid as an endonuclease, resulting in oligonucleotides with 3'-hydroxyl termini. The properties of this enzyme were distinct from those of the rat liver cytosol enzyme reported by Roewekamp and Sekeris in many respects, such as molecular weight, optimal pH and requirements for divalent cations. Preliminary experiments suggest that the nuclear enzyme is localized in the nucleoplasm and nucleoli. These results indicate that multiple forms of ribonuclease H exist in different regions of rat liver cells.     

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 3-methyladenine DNA glycosylase I from Escherichia coli.

We have purified 3-methyladenine DNA glycosylase I from Escherichia coli to apparent physical homogeneity. The enzyme preparation produced a single band of Mr 22,500 upon sodium dodecyl sulphate/polyacrylamide gel electrophoresis in good agreement with the molecular weight deduced from the nucleotide sequence of the tag gene (Steinum, A.-L. and Seeberg, E. (1986) Nucl. Acids Res. 14, 3763-3772). HPLC confirmed that the only detectable alkylation product released from (3H)dimethyl sulphate treated DNA was 3-methyladenine. The DNA glycosylase activity showed a broad pH optimum between 6 and 8.5, and no activity below pH 5 and above pH 10. MgSO4, CaCl2 and MnCl2 stimulated enzyme activity, whereas ZnSO4 and FeCl3 inhibited the enzyme at 2 mM concentration. The enzyme was stimulated by caffeine, adenine and 3-methylguanine, and inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide and 3-methyladenine. The enzyme showed no detectable endonuclease activity on native, depurinated or alkylated plasmid DNA. However, apurinic sites were introduced in alkylated DNA as judged from the strand breaks formed by mixtures of the tag enzyme and the bacteriophage T4 denV enzyme which has apurinic/apyrimidinic endonuclease activity. It was calculated that wild-type E. coli contains approximately 200 molecules per cell of 3-methyladenine DNA glycosylase I.     

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.     

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

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

Isolation, characterization, and activation of the magnesium dependent endodeoxyribonuclease from Bacillus subtilis.

A major endodeoxyribonulcease was isolated from a mutant of the transformable Bacillus subtilis 168. The magnesium-dependent endonuclease was purified approximately 750-fold to electrophoretic homogeneity. The enzyme had a molecular weight of about 31 000, as determined by gel filtration and polyacrylamide gel electrophoresis. The protein appears to be composed of two subunits. The nuclease was dependent on magnesium or maganese ions for hydrolytic activity. The purified nuclease degraded DNA from several species of Bacillus, as well as Escherichia coli DNA, alkylated, depurinated, and thymine-dimer containing B. subtilis DNA, and hydroxymethyluracil-containing phage DNA. The enzyme also hydrolyzed single-stranded DNA, although native DNA was the preferred substrate. However, the nuclease was unable to degrade ribosomal RNA. The cleavage products of the DNA hydrolysis have 5'-phosphate and 3'-hydroxyl ends. The enzyme could be activated in crude extracts by heat treatment or treatment with guanidine hydrochloride. The nuclease activity was inhibited by phosphate and by high concentrations of NaCl. A possible function for this endonuclease in bacterial transformation is discussed.     

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.     

5' leads to 3'-Exonucleases of bacteriophage T4.

Two enzyme activities which release nucleotides preferentially from the 5' termini of DNA were found in T4-infected Escherichia coli. Since no corresponding activities were found in uninfected cells, the activities appeared to be induced by T4. Both activities are capable of excising pyrimidine dimers from ultraviolet-irradiated DNA which has been treated with T4 endonuclease V. One of the activities , referred to as T4 exonuclease B, was purified 400-fold from an extract of T4v 1- infected cells. The enzyme initiates hydrolysis of DNA specifically at the 5' termini to yield products which are mainly oligonucleotides of varying length. The hydrolysis reaction proceeds in a limited manner. The enzyme shows optimal activity at pH 7.0 and absolutely requires Mg2+. The molecular weight of the enzyme , as estimated by gel filtration, is approximately 35,000. Another activity, referred to as T4 exonuclease C, was purified 240-fold from the extract. This activity also excises pyrimidine dimers from ultraviolet-irradiated, incised DNA and releases nucleotides at 5' termini. It has a pH optimum at 7.5 and requires Mg2+. The molecular weight of the enzyme is approximately 20,000.     

Drosophila apurinic/apyrimidinic DNA endonucleases. Characterization of mechanism of action and demonstration of a novel type of enzyme activity

Two species of apurinic/apyrimidinic (AP) endonuclease have been purified approximately 400-fold from extracts of Drosophila embryos. AP endonuclease I, which flows through phosphocellulose columns, has an apparent subunit molecular weight of 66,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas AP endonuclease II, which is retained by phosphocellulose, has a subunit molecular weight of 63,000. The molecular weight determinations were made possible in part by the finding that both Drosophila enzymes, along with Escherichia coli endonuclease IV, cross-react with an antibody prepared toward a human AP endonuclease (Kane, C. M., and Linn, S. (1981) J. Biol. Chem. 256, 3405-3414). The nature of phosphodiester bond breaks produced by the two partially purified AP endonucleases from Drosophila have been investigated. Nicks introduced into partially depurinated PM2 DNA by Drosophila AP endonuclease I did not support DNA synthesis by E. coli DNA polymerase I, whereas nicks created by AP endonuclease II were able to support DNA synthesis, but at a rate far less than that observed for nicks introduced by E. coli endonuclease IV. The priming activity of DNA incised by either of the Drosophila enzymes can be enhanced, however, by an additional incubation with E. coli endonuclease IV, which is known to cleave depurinated DNA on the 5'-side of an apurinic site. These results suggest that the Drosophila enzymes cleave depurinated DNA on the 3'-side of the apurinic site. This suggestion was strengthened by the observation that the combined action of AP endonuclease II and E. coli endonuclease IV resulted in the removal of [32P]dAMP from partially depyrimidinated [dAMP-5'-32P,uracil-3H]poly(dA-dT). Taken together, these results propose that Drosophila AP endonuclease II produces 3'-deoxyribose and 5'-phosphomonoester nucleotide termini. Conversely, the absolute inability to detect priming activity for DNA cleaved by AP endonuclease I alone suggested a different mechanism, possibly the formation of a deoxyribose-3'-phosphate terminus. When apurinic DNA cleaved by AP endonuclease I was subsequently treated with bacterial alkaline phosphatase, DNA synthesis was now detected at levels similar to that observed for AP endonuclease II alone. Additionally, DNA nicked by AP endonuclease I was susceptible to 5'-end labeling by polynucleotide T4 kinase without prior phosphomonoesterase treatment. These results suggest that AP endonuclease I forms deoxyribose 3'-phosphate and 5'-OH termini upon cleaving depurinated DNA.     

A deoxyribonuclease of Diplococcus pneumoniae specific for methylated DNA.

A deoxyribonuclease specific for methylated DNA was isolated from Diplococcus pneumoniae. The enzyme, an endonuclease, degrades DNA for Escherichia coli to fragments of average molecular weight about half a million; it forms discrete fragments from phage lambda DNA. Methyl-deficient E. coli DNA is not attacked, neither is DNA from Micrococcus radiodurans, which contains no methylated adenine or cytosine. Nor is DNA from D. pneumoniae or phage T7 attacked. However, DNA from M. radiodurans, D. pneumoniae, and T7 is attacked after methylation with and E. coli extract. Methylated T7 DNA is degraded to discrete fragments. Although the genetic transforming activity of normal DNA from D. pneumoniae is not affected by the enzyme, transforming activity of methylated DNA is destroyed. The enzyme is designated endonuclease R Dpn I. Under certain conditions another enzyme of complementary specificity can be isolated. This enzyme, designated endonuclease R Dpn II, produces a similar pattern of fragments from the DNA of T7 without prior methylation of the DNA. It also degrades normal DNA for D. pneumoniae. It is suggested that this pair of enzymes plays a role in some unknown control process, which would involve a large fraction of the specific base sequences that are methylated in E. coli DNA and are present but not methylated in DNA from other sources.