Snake Venom Exonuclease Research Articles

Single-strand-specific DNase activity is an inherent property of the 140-kDa protein of the snake venom exonuclease

Polyclonal antibodies against the exonuclease from Crotalus adamanteus venom (the 140-kDa protein) inhibit both the exonucleolytic and the single-strand-specific endonucleolytic activities, present in the exonuclease preparation. The antibodies also diminish the ability of the enzyme to split the negatively supercoiled Bluescript KS + in the AT-rich fragment near-by the transcription termination site of the Ampicillin gene. Therefore the single-strand-specific endonucleolytic activity was attributed to the protein molecule of the exonuclease. The processivity of the exonucleolytic action was found to be less than 3 monomers as indicated by the heparin trapping method.

Cleavage properties of an estrogen-regulated polysomal ribonuclease involved in the destabilization of albumin mRNA.

Previous work from this laboratory [Dompenciel,R.E., Garnepudi,V.R. and Schoenberg,D.R. (1995)J. Biol. Chem.270, 6108-6118] described the purification and properties of an estrogen-regulated endonuclease isolated from Xenopus liver polysomes that is involved in the destabilization of albumin mRNA. The present study mapped cleavages made by this enzyme onto the secondary structure of the portion of albumin mRNA bearing the major cleavage sites. The predominant cleavages occur in the overlapping APyrUGA sequence AUUGACUGA present in a single-stranded loop region, and in AUUGA located within a bulged AU-rich stem. A structural mutation which converted the major loop cleavage site to a hairpin bearing one APyrUGA element eliminated cleavage at the intact site. This confirms that the polysomal RNase is specific for single-stranded RNA. Additional point mutations in the major loop characterized the nucleoside sequence requirements for cleavage. Finally, snake venom exonuclease was used to demonstrate the polysomal RNase generates products with a 3' hydroxyl. Binding of an estrogen-induced protein to a portion of the 3'UTR of vitellogenin mRNA may be involved in its stabilization by estrogen [Dodson,R.E. and Shapiro,D.J. (1994)Mol. Cell. Biol.14, 3130-3138]. The core binding site for this protein bears the sequence APyrUGA, suggesting that stabilization may be accomplished by occlusion of a cleavage site for the polysomal RNase.

Synthesis of phosphonylmethyl analogues of diribonucleoside monophosphates containing modified internucleotide bond

Two types of (2'-5')- and (3'-5')-isomers of analogues of diribonucleoside monophosphates derived from O-phosphonylmethyl derivatives of ribonucleosides, differing in the position of the methylene group in the internucleotide bond (type A, B, C, and D) have been synthesized. The compounds were prepared from methyl esters of O-phosphonylmethylribonucleosides I and XVII by a procedure analogous to the phosphotriester method of oligonucleotide synthesis. The phosphonate moiety was protected with the methyl group. After protection of the hydroxyl or amino groups, the compounds I or XVII were condensed with protected ribonucleosides VIII, XI, XIV, XXIII to afford the neutral diesters IX, XII, XV, XXIV, XXVI, and XXVIII which were isolated by short column chromatography on silica gel. Deprotection, ion-exchange chromatography, and semipreparative HPLC gave (2'-5')- and (3'-5')-isomers of both types of O-phosphonylmethyl analogues of diribonucleoside monophosphates (X, XIII and XXV, XXVII). All these compounds are resistant towards cleavage with ribonucleases A and T2 and with snake venom exonuclease. Under conditions of alkaline hydrolysis of RNA, the analogues of the type A and B are completely stable whereas compounds of the type C and D are degraded to form 2'- or 3'-O-phosphonylmethylribonucleosides and 3'-terminal ribonucleosides.

Detection of neocarzinostatin chromophore-deoxyribose adducts as exonuclease-resistant sites in defined-sequence DNA.

A 5'-end-labeled DNA restriction fragment was treated with the nonprotein chromophore of neocarzinostatin under anoxia in the presence of dithiothreitol, conditions known to maximize formation of chromophore-deoxyribose adducts. Under conditions where unmodified DNA was digested to completion, chromophore-treated DNA was highly resistant to digestion by exonuclease III plus the 3'----5' exonucleolytic activity of T4 DNA polymerase and partially resistant to digestion by exonuclease III plus snake venom exonuclease. The electrophoretic mobilities of the products of exonucleolytic digestion suggested that (i) digestion by exonuclease III or T4 polymerase terminated one nucleotide before the nucleotide containing the adduct, (ii) the remaining nucleotide directly adjacent to the adduct (3' side) could be removed by snake venom phosphodiesterase, but at a slow rate, (iii) the covalently linked chromophore decreased the electrophoretic mobilities of the digestion products by the equivalent of approximately three nucleotides, and (iv) adducts formed under anaerobic conditions occurred at the same nucleotide positions as the strand breaks formed under aerobic conditions (primarily at T and, to a lesser extent, A residues). The close similarity in sequence specificity of adducts and strand breaks suggests that a common form of nascent DNA damage may be a precursor to both lesions. A chromophore-induced free radical on C-5' of deoxyribose, subject to competitive fixation by addition reactions with either oxygen or chromophore, is the most likely candidate for such a precursor. The base specificity of adduct formation does not reflect the reported base specificity of neocarzinostatin-induced mutagenesis, suggesting that lesions other than adducts may be responsible for at least some neocarzinostatin-induced mutations, particularly those occurring at G X C base pairs.

Nucleotide sequence of polypyrimidines from cloned mouse DNA as determined by base-specific blockage of exonuclease action

Cloned fragments of mouse DNA have been screened for the presence of long polypyrimidine/polypurine segments. The polypyrimidine portion of one such segment (about 200 nucleotides in length) has been isolated by acidic depurination of the entire cloned fragment and plasmid vector followed by selective precipitation and 5′- 32P labeling. This polypyrimidine has been used to demonstrate a new procedure for sequencing. Covalent modification of thymine with a water-soluble carbodiimide, or cytosine with glutaric anhydride, at low levels blocked the action of snake venom exonuclease. After deblocking, separation of the products of digestion by polyacrylamide gel electrophoresis yields a sequence ladder which can be used to determine the position of C and T residues as in other sequencing methods. A sequence of 72 residues adjacent to the 5′ end has been established, consisting principally of the repeating tetranucleotide (CCTT) n. A low ratio of endonuclease to exonuclease is essential for application of this method to sequences of this size. Accordingly, a very sensitive modification of a fluorometric endonuclease assay was developed and used to optimize pH and Mg 2+ conditions to favor exonuclease activity over the accompanying endonuclease activity. The results clearly indicate that long polypyrimidine tracts can be efficiently prepared and their sequences determined with this method using commercially available exonuclease preparations without additional purification.

Neocarzinostatin chromophore-DNA adducts: evidence for a covalent linkage to the oxidized C-5' of deoxyribose.

The nonprotein chromophore of neocarzinostatin forms a variety of adducts with DNA. The predominant adduct recovered from nuclease digests of chromophore-treated poly(dA-dT). poly(dA-dT) is a compound with structure chromophore-d(TpApT). Mild acid hydrolysis of this compound released free adenine, while snake venom exonuclease (pH 6.5) released 5'-dTMP leaving in both cases adducts of slightly altered chromatographic mobility. These results eliminate adenine and 5'-dTMP as possible sites of covalent chromophore attachment. Electrophoresis data suggest that the adduct is not a phosphotriester. At pH 8.6, chromophore-d(TpApT) spontaneously hydrolyzed, releasing chromophore and 3'-dTMP, leaving a modified d(ApT) which contained deoxyadenosine-5'-aldehyde. Deoxyadenosine-5'-aldehyde was released from the modified d(ApT) by snake venom exonuclease, and identified by a series of derivatizations including 1) mild oxidation to deoxyadenosine-5'-carboxylic acid, 2) NaBH4 reduction to deoxyadenosine, and 3) formation of a hydrazone with phenylhydrazine. Since deoxyadenosine-5'-aldehyde cannot exist as such in the chromophore-d(TpApT) adduct, we suggest that the chromophore may be covalently attached to the C-5' of deoxyadenosine as a phosphorylacetal or similar structure. Hydrolysis of the chromophore-acetal bond at pH 8.6 would leave a phosphorylhemiacetal on C-5', which would be expected to spontaneously decompose to yield the observed 3'-phosphate and 5'-aldehyde groups.

Neutral deoxyribonucleases of HeLa S3 cells: electrophoretic separation, characterization, substrate specificity and mode of action.

Extracts of HeLa S3 cells were electrophoresed on polyacrylamide gels; gel slices were eluted and the eluates were assayed for DNase activities against native and denatured DNA substrates in the presence of MgCl2 or Na2EDTA. Aliquots of each eluate were also assayed for their ability to nick the circular supercoiled PM2 phage DNA to distinguish endonucleases from exonucleases. Peaks of endonuclease activities were characterized as forming 3'-phospho-oligonucleotides or 5'-phospho-oligonucleotides by the use of oligonucleotides produced by these enzymes as substrates for the 5'-phosphate-specific snake venom exonuclease. The total activity of DNases in gel eluates was much higher than that in cell extract applied to the gel, indicating the presence of inhibitors in the cell extract.

Diastereomers of 5'-O-adenosyl 3'-O-uridyl phosphorothioate: chemical synthesis and enzymic properties

A procedure is described for the synthesis of the title compounds via phosphotriester intermediates. The 2-cyanoethyl group is used to protect the P-SH function during the course of the synthesis. Resolution of the phosphorus diastereomers is accomplished at the phosphotriester stage. Removal of the 2-cyanoethyl group without racemization, followed by removal of the other protective groups, affords the optically pure diastereomers of 5'-O-adenosyl 3'-O-uridyl phosphorothioate. Their designation as Rp and Sp follows from the stereospecificity in the hydrolysis catalyzed by RNase A. These diastereomers are useful for the investigation of the stereospecificity as well as of the stereochemical course of action of nucleases. Snake venom exonuclease hydrolyses only the Rp diastereomer, whereas both diastereomers are substrates for RNases A and T2. The results with the latter indicate that RNase T2 also operates by an in-line mechanism.

Mapping of deoxydi- and trinucleotides liberated by the action of endonucleases from the silkworm and Aspergillus oryzae

Both silkworm nuclease and nuclease O of Aspergillus oryzae mycelia hydrolyse DNA endolytically to di- and trinucleotides terminating in 5′-phosphate. These oligonucleotides were fractionated first by DEAE-cellulose chromatography with 7 M urea into the respective isoplithic groups and then analysed for the composition and isomerism: each group was labeled 5′-terminally by the [γ- 32P]ATP-polynucleotide kinase reaction and then electrophoresed monodimensionally for the dinucleotides and two-dimensionally for the di- and trinucleotides mixtures, respectively, followed by elution and digestion with snake venom exonuclease. Both nucleases gave rather similar simple maps in which all sixteen dinucleotides and almost all the possible trinucleotides were identified, indicating their random mode of actions.

Control of breakdown of the polyadenylate sequence in mammalian polyribosomes: role of poly(adenylic acid)-protein interactions.

The poly(adenylic acid) [poly (A)] segment in mouse sarcoma polysomes in not hydrolyzed by snake venom exonuclease under conditions which cause extensive degradation of the poly(A) in deproteinized polysomal RNA. The protecting effect of polysomes is presumably caused by the interaction between the poly(A) sequence and the protein known to be associated with it. This protection is reduced at low KCl concentration, but addition of exogenous RNA restores the protecting effect. The poly(A) segment also becomes susceptible to exonuclease after fragmentation of the polysomes by mild ribonuclease treatment. The latter treatment releases the poly(A) in association with protein. The poly(A) sequence in polysomes in readily degraded by a cytoplasmic extract of S-180 cells. Partial purification leads to a preparation active against the poly(A) in polysomes under conditions where no fragmentation of the messenger RNA is observed. Snake venom exonuclease increases the activity of the cytoplasmic preparation against poly(A) in polysomes. The active cytoplasmic factor appears to interfere with the poly(A)-protein interaction, thus rendering the polynucleotide susceptible to degradation by exonuclease. The poly(A) sequences in polysomes and in free cytoplasmic nucleoprotein particles are hydrolyzed to the same extent. The results suggest that the poly(A) sequence is normally protected from nucleases by virtue of its association with protein. The slow reduction in poly(A) size in cytoplasmic mRNA can be accounted for by a factor capable of interfering with the poly(A)-protein interaction. The latter interaction seems also dependent on the structural integrity of the polysomes or messenger ribonucleoproteins. It is suggested that a polynucleotide segment adjacent to the poly(A) can modulate the affinity of the protein for the latter sequence, thus permitting control of poly(A) stability in individual messenger RNAs.

Nucleotide sequence of the restriction fragment Hind F - Eco RI2 of SV40 DNA

The nucleotide sequence of the SV40 genome region between the Hind K fragment and the Eco RI cleavage site has been determined by a combination of three different approaches : analysis of RNA products obtained by transcription with Escherichia coli DNA dependent RNA polymerase, partial degradations with snake venom exonuclease and base-specific chemical degradation of 5'-terminal labeled restriction fragments. This nucleotide sequence shows only one open reading frame and allows the deduction of a small segment of the amino acid sequence of VP1, the major structural protein.

Sequence relationships between the genome and the intracellular RNA species of standard and defective-interfering semliki forest virus

Oligonucleotide fingerprinting on slabs of polyacrylamide gel (De Wachter & Fiers, 1972) has been used to investigate the sequence relationships between the 42 S RNA genome of standard Semliki Forest virus and the intracellular virus-specified single-stranded RNAs isolated from baby hampster kidney cells infected with standard virus and co-infected with standard virus and defective-interfering particles. For the intracellular RNAs of standard virus-infected cells these studies show that the nucleotide sequence of the 26 S RNA, which acts as messenger RNA for the structural proteins of the virus particle Clegg and Kennedy, 1975a , Clegg and Kennedy, 1975b , is located at the 3′ end of the 42 S RNA and that the 38 S and 33 S RNAs which appear as minor species on RNA polyacrylamide gels run under non-denaturing conditions, are conformational variants of the 42 S and 26 S RNAs, respectively. Studies on the two RNAs of defective-interfering particles isolated from cells co-infected with standard virus and defective-interfering particles indicate extensive sequence homology between the defective-interfering RNAs, and show that both RNAs contain nucleotide sequences present in both the structural and non-structural genes of the 42 S RNA. Furthermore, by determining the order (5′→3′) of the characteristic oligonucleotides of the 42 S RNA, and by digestion with snake venom exonuclease, it is concluded that the nucleotide sequences of the defective-interfering RNAs are derived from the 5′ and 3′ extremities of the 42 S RNA; about 75% from the 5′ end and 25% from the 3′ end. The significance of this observation in relation to the genesis of defective-interfering particles and to the mechanism of interference by the defective-interfering RNAs is discussed.

Partial degradation of transfer RNAs by different preparations of snake venom exonuclease

The degradation of yeast tRNA Ser with eight different exonuclease preparations from four snake venoms was investigated. The reaction products were separated on polyacrylamide gels containing 7 M urea. Patterns of sharp bands were obtained which were more or less similar. Two tRNA fragments were characterized by oligonucleotide analyses, one of which was tRNA degraded by the exonuclease up to the beginning of the T-ψ-C-stem. The other one was generated by the additional loss of several nucleotides from the 5′-terminus. The formation of the latter fragment was very probably caused by an endonuclease activity in the exonuclease. The endonuclease contaminant, which was found in all preparations, was further investigated by experiments with modified tRNAs whose 3′-terminus should be resistant to exonuclease (tRNA Ser -A, tRNA Ser ox-red). With 3′-AMP as substrate no phosphatase activity was found under the conditions of tRNA degradation. Not only in tRNA Ser, but also in yeast tRNA Tyr and tRNA Ala as well as in fragments of tRNA Ser and tRNA Phe, the degradation by exonuclease was inhibited at the beginning of the T-ψ-C-stem. The finding of such a retardation site in addition to the general retardation of exonuclease digestion after removal of the C-C-A sequence may indicate that retardation at certain elements of secondary structure is a more general feature of degradations by this enzyme.

A fast method for the determination of 5′ terminal dinucleotides of DNA fragments

A method for the rapid analysis of 5′ dinucleotides of DNA fragments is described. The method involves: (a) Labeling of 5′ termini with polynucleotide kinase, (b) digestion of the oligonucleotides to dinucleotides with pancreatic DNAase and exonuclease I from Escherichia coli: (c) unidimensional separation by electrophoresis of all the dinucleotides with the exception of the isomers: (d) elution and further hydrolysis of the isomers to 5′ mononucleotides with snake venom exonuclease: and (e) unidimensional separation by thin-layer chromatography of the four 5′ nucleotides.

Poly(U) in poliovirus minus RNA is 5′-terminal

Summary Poly(U) isolated from polio [32P]RF has the base composition (U65, C2)Gp. Labeling of the poly(U) in RF with NaIO4/[3H]NaBH4 failed, even if the RF was pretreated with phosphatase. Labeling of poly(U) in RF with [γ-32P]ATP and polynucleotide kinase occurs but only after treatment of the RF with either snake venom exonuclease or polynucleotide phosphorylase. The data suggest that poly(U) is the 5′-end of polio minus RNA.

A fast method of analysis of the 5′ terminal nucleotides of deoxyribooligonucleotides

A method for the rapid analysis of the 5′ ends of deoxyribooligonucleotides is described. The method involves: (a) labeling of the 5′ ends with polynucleotide kinase; (b) digestion of the oligonucleotides to 5′ mononucleotides with pancreatic DNAase and snake venom exonuclease; (c) unidimensional separation on polyethyleneimine plates of the four 5′ mononucleotides, ATP, and inorganic phosphate. This method requires only a minimum amount of work, time, and material.

A new kinetic model for polynucleotide metabolism.

Several exonucleases, such as the exoribonuclease from Ehrlich asciltes tumor cells,1 Escherichia coli ribonuclease II,2 and bacterial polynucleotide phosphorylase,3 4 have recently been showrn to remain complexed with an individual polyriucleotide molecule while the enzyme continuously and almost completely degrades the polymer to mononucleotides. The behavior of these exonucleases is in marked conitrast to that of the exonuclease from snake venom2' I and exonuclease I from E. coli.5 Wheni snake venom exonuclease hydrolyzes polynucleotides, the enzyme-substrate complex dissociates into separate entities between successive hydrolytic steps, and the next phosphodiester bond hydrolyzed comes from a polynucleotide molecule taken at random from the molecules in the vicinity of the enzyme. However, when Ehrlich ascites tumor cell exoribonuclease, E. coli ribonuclease II, and bacterial polynucleotide phosphorylase degrade polynucleotides, the polynucleotide apparently does not dissociate from the enzyme after cleavage of the terminal mononucleotide, but rather the enzyme and shortened polyniucleotide move relative to each other so as to bring the next phosphodiester bond into position for cleavage. By means of such processiont, ail entire polynucleotide molecule may be degraded without ever leaving the surface of the enzyme. The presumption of a processive step implies that after a hydrolytic step, the enzyme and shortened polynucleotide are still held together by one or more bonds. We wish to point out that the processes of dissociatioin and processioni of the polynucleotide should not be considered as mutually exclusive; after the first cleavage step, there will in general be a probability for dissociatiorn and a probability for procession, the values for which are related to the rate constanits for the two processes. We would like to present, a formal kinetic model of exonucleolytic action that itteludes both classes of exonucleases as special cases for limiting values of the Uvilctic constants. This model has beeni used to design kitnetic experimeiits wvith tih exoriboiiuclease from Ehr lich ascites tumor cells. The data yield iriforilatioul about: (1) the number of bonids between the enzyme and the polyinucleotide beinig degraded; (2) the energetic contribution of each bond; and (3) the freeenergy drop causinig procession of the polynucleotide on the enzyme, which in turn allows this particular enzyme to degrade a polynucleotide in a continuous mannier. Although the kinetic model is applicable to both random and continuous exonucleolytic degradation, it will be presented and illustrated (Fig. 1) with emphasis on the special case of continuous degradationi: The enzyme is assumed to bind a substrate, such as polyadenylic acid (Poly A), of chain length n (step 1, Fiig. 1). The binding is assumiied to be between rn filled binditig places on the enzyme and m places on the polymer. The chain length, n, of the polymer is greater than the number of binding places, rn. The hydrolysis of the terminal