Purification of a Nuclease from Penicillium citrinum

A nuclease was purified about 1500-fold with a recovery of 20% from an aqueous extract of culture of a pigmentless mutant VI–10–14 of Penicillium citrinum on wheat bran. The purified preparation was homogeneous on the basis of the criteria of ultracentrifugation and disc gel electrophoresis. The preparation was essentially free of 5′-nucleotidase, non-specific phosphomonoesterase, non-specific phosphodiesterase and 3′-monoester forming nuclease. The preparation hydrolyzed phosphodiester bonds in RNA and DNA to yield 5′-mononucleotides, and also the phosphomonoester bond in 2′- and 3′-AMP to yield nucleoside and inorganic phosphate. The enzyme activities toward these substrates were not separated and relative ratio of their specific activities remained constant throughout the purification, suggesting that a single enzyme was responsible for these activities.     

Purification and properties of a light-inducible nuclease from Euglena gracilis.

The nuclease described by Carell, E.F., Egan, J.M. and Pratt, E.A. [Arch. Biochem. Biophys. (1970) 138, 26-31] has been purified 1000-fold from Euglena gracilis strain Z. The enzyme catalyzes the hydrolysis of both polyribonucleotides and polydeoxyribonucleotides. The relative rates of hydrolysis of synthetic and natural polynucleotides was found to be: poly (U) 100, poly (dT) 33, denatured calf-thymus DNA 33, yeast tRNA 9, E. coli total RNA 6, poly (dA dT) 5, poly (A) less than 1, poly (C) less than .05, and poly (G) less than .05. The enzyme attacks polynucleotides in an endonucleolytic fashion, yielding products terminated with a 3'-phosphate. Poly (U) appears to be hydrolyzed completely to 3'-UMP; both RNA and DNA appear to have some phosphodiester bonds resistant to enzyme catalyzed hydrolysis. Because of its mode of action and its inducibility by light, we propose the name endonuclease L for this enzyme.     

Constituents of Plant Leaf Wax Contained in Rice Callus Tissues

Two enzyme preparations having both nuclease and 3′-nucleotidase activities were partially purified from an extract of tea leaves. They resemble each other in most enzymatic properties, but are separated by DEAE-cellulose column chromatography.

The enzyme activities for RNA, native DNA, heat-denatured DNA and 3′-AMP of each preparation showed a high degree of similarity with respect to the following properties: pH stability, thermal stability and response to EDTA. Both enzymes were shown to be endonucleases (EC 3.1.30.2) which liberated 5′-mononucleotides and oligonucleotides from both RNA and DNA with the following relative rate of hydrolysis: RNA > native DNA = heat-denatured DNA.     

Purification and characterization of a major endonuclease from rat liver nuclei.

A major endonuclease has been purified approximately 800-fold from rat liver nuclei using poly(A) as substrate. The enzyme had a molecular weight of about 50,000, and active fractions were obtained which contained no nucleic acid. Enzymatic activity was optimal between pH 6 and 7 and was totally dependent on the presence of a divalent cation. The reaction was inhibited by high ionic strength, polydextran sulfate, heparin, and sodium pyrophosphate. The purified enzyme readily hydrolyzed poly(A), poly(U), poly(C), and denatured DNA, whereas poly(G) was not degraded, and transfer RNA, ribosomal RNA, and native DNA were hydrolyzed only at relatively slow rates. These data suggest that the enzyme may be specific for single-stranded polynucleotides. The purified enzyme was essentially devoid of exonuclease activity, and the products of exhaustive endonuclease digestion of poly(A) were small oligonucleotides terminated with a 5'-phosphoryl group. Evidence was obtained that this endonuclease is localized in the nucleoplasm. Possible functions for this activity are discussed.     

An Extracellullar Nuclease from Bacillus Firmus VKPACU-1: Specificity and Mode of Action

An extracellular nuclease from Bacillus firmus VKPACU-1 was multifunctional enzyme, this nuclease hydrolyzed poly U rapidly and more preferentially than the other homopolyribonucleotides. Hydrolysis of RNA this enzyme released mononucleotides in the order 5′UMP > 5′AMP > 5′GMP where as in hydrolysis of DNA the mononucleotides in the order of 5′dAMP > 5′dGMP > 5′dTMP and oligonucleotides. Uridylic linkages in RNA and adenylic linkages in DNA were preferentially cleaved by the nuclease. Nuclease produced oligonucleotides having only 3’ hydroxyl and 5’ phosphate termini. Present nuclease hydrolyzed RNA and DNA released oligonucleotides as major end products and mononucleotides, suggesting an endo mode of action.     

Substrate specificity of Ca2+,Mg2+-dependent DNAase from sea urchin (Strongylocentrotus intermedius) embryos

Ca2+,Mg2+-dependent DNAse from sea urchin embryos is specific to the secondary structure of substrates irrespective of the nature of activating cations. The enzyme does not split synthetic single-stranded oligo and polynucleotides, such as d(pTpTpTpCpC), d(pGpGpTpTpT). d(pApApTpTpC), d(pGpApApTpTpC), d(pA)5-poly(dT), d(pApApTpTpC)-poly(dT), poly(dA) and poly (dT) and hydrolyses the double-stranded substrates poly d(AT), poly (dA) . poly (dT) and highly polymerized DNA. Native double-stranded DNA from salmon and phage T7 is split by the enzyme at a higher rate than that of denaturated DNA of salmon and single-stranded DNA of phage M13. The high rate of poly(dA) . poly(dT) and poly d(AT) hydrolysis and the stability of poly(dG) . poly(dC) to the effect of the enzyme suggest a certain specificity of the enzyme to the nature of nitrogenous bases at the hydrolyzed phosphodiester bond of the substrate.     

Carcinogenic purine N-oxide ester modifies covalently all common bases in polynucleotides

The carcinogen 1-methyl-3-hydroxyxanthine after esterification binds covalently to polynucleotides, RNA and DNA. All four ribopolynucleotides and poly(dT) are targets. Depending on reaction conditions, covalent binding is greatest to poly(A) followed by poly(U), poly(dT), poly(G), poly(C), RNA and DNA. Maximal covalent modification of DNA is one moiety per 360 nucleotides. All modified polynucleotides, RNA and DNA, except poly guanylic acid have been enzymatically digested and the major adducts characterized as nucleosides.     

Purification and some properties of a nuclease from rye germ nuclei

1. An endonuclease has been isolated from the nuclei of rye (Secale cereale L) germ and partially purified. The enzyme shows optimum activity over the pH range 5.4-7.4 towards both DNA and RNA, and has no phosphomonoesterase or phosphodiesterase activity. 2. DNA is degraded by the rye germ nuclease to oligonucleotides of similar size, and RNA to oligonucleotides and mononucleotides containing a C-terminal 5'-phosphate group. 3. The rate of hydrolysis of nuclear acids by the enzyme decreases in the following order: native DNA greater than denatured DNA greater than RNA. Synthetic polynucleotides are hydrolysed at a rate decreasing in the order: poly(A) greater than poly(U) greater than poly(C) greater than poly(G).     

Differential susceptibilities of DNA polymerases-alpha and -beta to polyanions.

The effects of various polyanions including synthetic polynucleotides on DNApolymerases-alpha and -beta from blastulae of the sea urchin Hemicentrotus pulcherrimus and HeLa cells were studied. Only DNA polymerase-alpha was inhibited by polyanions, such as polyvinyl sufate, dextran sulfate, heparin, poly(G), poly(I), poly(U) and poly(ADP-Rib). Of the various polynucleotides tested, poly(G) and poly(I) were the strongest inhibitors. Kinetic studies showed that the Ki value for poly(G) was 0.3 microgram/ml and that poly(G) had 20-fold higher affinity than activated DNA for the template-primer site of DNA polymerase-alpha. Poly(U) and poly(ADP-Rib) were also inhibitory, but they were one hundredth as inhibitory as poly(G) or poly(I). Poly(A), poly(C), poly(A).poly(U) AND POLY(I).poly(C) were not inhibitory to DNA polymerase-alpha. In contrast, DNA olymerase-beta was not affected at all by these polyanions under the same conditions.     

S1 nuclease of Aspergillus oryzae: a glycoprotein with an associated nucleotidase activity

S₁ nuclease (EC 3.1.30.1) of Aspergillus oryzae has been purified 1600-fold by a procedure designed to remove traces of contaminating phosphatases. The nearly homogeneous enzyme was found to be a glycoprotein with a carbohydrate content of 18%. At pH 4.5 the enzyme preparation hydrolyzed single-stranded DNA, RNA, 3′-AMP, and 2′-AMP at relative rates of 100, 52, 13, and 0.05, respectively. The 3′-nucleotidase activity of this single-strand specific nuclease is inhibited by single-stranded DNA but not by double-stranded DNA. Three forms of the enzyme, with isoelectric points of 3.35, 3.53, and 3.67, were observed on electrofocusing, and each form exhibited the same relative activity on single-stranded DNA and 3′-AMP. Enzymatic hydrolysis of nucleotides occurred over a broad range of pH, with maximal activity at pH 6–7. Ribonucleotides were hydrolyzed approximately 100-fold more rapidly than deoxyribonucleotides. A high degree of base specificity was not observed. The 3′-nucleotidase activity was stimulated by Zn²⁺, but not by other divalent cations tested.     

Distribution of P1 Type Nuclease in the Genus Penicillium

P₁ type nuclease, which hydrolyzes RNA and heat-denatured DNA completely into 5’-mononucleotides and also shows 3’-nucleotidase activity, was widely distributed among various species belonging to the genus Penicillium such as P. expansum, P. notatum, P. steckii and P. meleagrinum. P₁ type nucleases isolated from these strains were produced in a form of complex with malonogalactan when molds were grown on wheat bran. These enzymes showed similar characters in heat-stability (stable at 60°C), temperature optimum (60 to 70°C for RNA and heat denatured DNA, and 70°C for 3’-AMP) and sensitivity to EDTA. The enzymes from P. steckii and P. expansum were immunologically co-related to nuclease P₁.

In addition, many strains of Penicillium produced base-nonspecific RNases forming 3’-mononucleotides via 2’: 3 ’-cyclic nucleotides. These RNases showed similarity in heat-lability (completely inactivated at 60°C), temperature optimum (45 to 50°C), sensitivity to Zn²⁺ and Cu²⁺, and relative hydrolysis rate toward 2’: 3’-cyclic nucleotides (A?C>U?G).     

DNA methylation. Inhibition of de novo and maintenance methylation in vitro by RNA and synthetic polynucleotides

A partially purified HeLa cell DNA methylase will methylate a totally unmethylated DNA (de novo methylation) at about 3-4% the rate it will methylate a hemimethylated DNA template (maintenance methylation). Our evidence suggests that many, if not most, dCpdG sequences in a natural or synthetic DNA can be methylated by the enzyme. There is a powerful inhibitor of DNA methylase activity in crude extracts which has been identified as RNA. The inhibition of DNA methylase by RNA may indicate that this enzyme is regulated in vivo by the presence of RNA at specific chromosomal sites. The pattern of binding of RNA to DNA in the nucleosome structure and the DNA replication complex may determine specific sites of DNA methylation. An even more potent inhibition of DNA methylase activity is observed with poly(G), but not poly(C), poly(A), or poly(U). The only other synthetic polynucleotides studied which inhibit DNA methylation as well as poly(G) are the homopolymers poly(dC).poly(dG) and poly (dA).poly(dT). These results point out the unique importance of the guanine residue itself in the binding of the DNA methylase to dCpdG, the site of cytosine methylation. The surprising inhibition of the methylation reaction by poly(dA).poly(dT), which is itself not methylated by the enzyme, suggests the possible involvement of adjacent A and T residues in influencing the choice of sites of methylation by the enzyme.     

Purification and Some Properties of Plant Endonuclease from Scallion Bulbs

A plant endonuclease with 3′-nucleotidase activity was purified from scallion bulbs to homogeneity as judged by SDS-PAGE. The molecular weight of the enzyme was estimated to be 38,000 by SDS-PAGE and 40,000 by Sephadex G-100 gel filtration. The enzyme rapidly hydrolyzed yeast RNA and denatured calf thymus DNA to acid-soluble substances, and hydrolyzed the plasmid pBR322 to yield small DNA fragments at low enzyme concentrations. These four activities were eliminated by treatments with EDTA and tetraethylenepentamine. The enzyme had maximum activity at pH 8.5-9.0 for 3′-AMP, 3′-GMP, and 3′-UMP, at pH 6.5 for 3′-CMP and yeast RNA, and at pH 6.0 for denatured calf thymus DNA and pBR322. During digestion of yeast RNA by the enzyme at pH 6.5, 5′-GMP was released most rapidly, followed by 5′-UMP, 5′-AMP, and 5′-CMP. These properties were different from those of endonucleases isolated from other sources such as mung bean sprouts and wheat seedlings.     

Anti-complement activity of polynucleotides.

A biologic assay system, based on complement (C′) inhibition, is described to unravel structural differences among polynucleotides. The C′ system appears particularly suitable to distinguish (1) homo- from copoly-ribonucleotides, (2) deoxyribo- from 2′-OH and other 2′-modified polynucleotides, and (3) single homopolynucleotides from double- or triple-stranded complexes.From these studies a number of polynucleotides emerged with potent anti-C′ activities, worthy of further investigation. The most active polymers were (G)_n (polyguanylic acid), (dCc1)_n [poly(2′-chloro-2′deoxycytidylic acid)] and (dUz)_n [poly(2′-azido-2′-deoxyuridylic acid)].     

Human platelet ribonuclease

Human platelets contain an RNase which has a pH optimum at 5.0. It hydrolyzes the secondary phosphate esters of uridine 3′-phosphates. It slowly converts uridine 2′:3′-and cytidine 2′:3′-cyclic phosphates to their corresponding nucleoside 3′-phosphates. Poly (A), poly (G) and poly (C) are not only refractory to the action of this enzyme, but also inhibit its action on poly (U). It differs from human granulocyte RNase, human serum RNase and bovine pancreatic RNase. Because of its unique property, this enzyme could serve as a biochemical marker in disorders involving the platelet destruction.     

Three-step purification of retinol-binding protein from rat serum

An endoribonuclease has been purified about 320-fold from the microsomes of rat liver. The enzyme had an apparent molecular weight of 54 000-58 000 and produced oligonucleotides, each consisting of 3-7 nucleotides from poly(A) and poly(U). No mononucleotide was obtained by the enzymatic hydrolysis of poly(A) and poly(U) under standard coditions. The relative rates of breakdown of synthetic polynucleotides by the enzyme under standard conditions were in the order poly(U) = poly(A) > poly(C). Divalent cations (Mg2+ or Mn2+) was required for the enzymatic activity, but monovalent cations (Na+, K+ or NH4+) inhibited the enzyme. The breakdown of poly(C) and poly(U) by the enzyme was inhibited by spermine, but that of poly(A) was not influenced by spermine. The enzyme was inhibited by p-chloromercuribenzoate and poly(G), but not by rat-liver ribonuclease-inhibitor and anti-RNase A serum.     

Studies on the Autolysis of Aspergillus oryzae

Mode of action of crystalline nuclease O obtained from autolyzed Aspergillus oryzae on RNA and synthetic homopolymers was examined. Crystalline nuclease O had no strict base specificity, although the velocity of hydrolysis was poly A > poly U > RNA > poly C. This enzyme did not degrade poly G. Digestion of high molecular weight RNA with an excess of this enzyme produced mono-, di- and trinucleotides with 5′-terminal phosphate. The amount of mono-, di- and trinucleotides was, respectively, 13.6, 70.0 and 16.4% of total degradation products. All the four bases were detected in mononucleotide fraction and 3′-terminals and 5′-terminals of oligonucleotides.     

Purification and Some Properties of Nucleases from Shii-take,Lentinus edodes

Three kinds of nuclease preparations, each of which having both endonuclease activity that formed 5′-mononucleotides and 3′-nucleotidase activity, were separated and partially purified from Shii-take, Lentinus edodes. Both enzyme activities of each preparation showed a similar thermostability and electrophoretic mobility on Polyacrylamide gel, and a competitive relationship was observed between RNA and 3′-AMP in their enzyme reactions. From these results, it is concluded that both enzyme activities of these three preparations reside in a single protein, respectively. They resemble one another in substrate specificity, cleavage pattern of RNA and thermostability, but are distinguishable from one another by molecular weight, electrophoretic mobility and optimum pH for degradation of RNA.     

A 5'----3' exoribonuclease of Saccharomyces cerevisiae: size and novel substrate specificity

The purification scheme for a 5'----3' exoribonuclease of Saccharomyces cerevisiae has been modified to facilitate purification of larger amounts of enzyme and further extended to yield highly purified enzyme by use of poly(A)-agarose chromatography. As determined by either sodium dodecyl sulfate-polyacrylamide gel electrophoresis or physical characterization, the enzyme has a molecular weight of about 160,000. Further studies of its substrate specificity show that poly(C) and poly(U) preparations require 5' phosphorylation for activity and that poly(A) with a 5'-triphosphate end group is hydrolyzed at only 12% of the rate of poly(A) with a 5'-monophosphate end group. DNA is not hydrolyzed, but synthetic polydeoxyribonucleotides are strong competitive inhibitors of the hydrolysis of noncomplementary ribopolymers. Poly(A).poly(U) and poly(A).poly(dT) are hydrolyzed at 60 and 50%, respectively, of the rate of poly(A) at 37 degrees C. The RNase H activity of the enzyme can also be demonstrated using an RNA X M13 DNA hybrid as a substrate. When poly(dT).poly(dA) with a 5'-terminal poly(A) segment on the poly(dA) is used as a substrate, the enzyme hydrolyzes the poly(A) "tail," removing the last ribonucleotide, but does not hydrolyze the poly(dA).     

Adenylyl-(5′, 2′)-adenosine 5′-Phosphate ((2′-5′) pA-A) in Crude Crystals of 5′-Adenylic Acid Isolated from Nuclease P1 (Penicillium Nuclease) Digest of Technical Grade Yeast RNA

Adenylyl (5′,2′)-adenosine 5′-phosphate ((2′-5′)pA-A) was detected in crude crystals of 5′-AMP prepared from Penicillium nuclease (nuclease P₁) digest of a technical grade yeast RNA. While (3′–5′)A-A was split by nuclease P₁, spleen phosphodiesterase, snake venom phosphodiesterase or alkali, (2′–5′)A-A was not split by a usual level of nuclease P₁ or spleen phosphodiesterase. Nuclease P₁ digests of 12 preparations of technical grade yeast RNA tested were confirmed to contain (2′–5′)pA-A. Its content was about 1 to 2% of the AMP component of each RNA preparation. As poly(A) was degraded completely by the Penicillium enzyme into 5′-AMP without formation of any appreciable amount of (2′–5′)pA-A, the technical grade RNA is supposed to contain 2–5′ phosphodiester linkages in addition to 3′–5′ major linkages.