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Results for “Endodeoxyribonucleases”

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PubMed1986

Integration of transposon Tn10 into phage L (Salmonella typhimurium).

Transposon Tn10 was transposed into phage L (Salmonella typhimurium) from F'ts114lac+zzf::Tn10 plasmid of strain TT629 (Chumely et al. 1979). Phage L with the insertion Tn10 (L::Tn10-8) was isolated in the form of a prophage in the lysogenic strain S. typhimurium LT2-18 (L::Tn10-8), in which it can be induced with UV light. The phage induced in this way is defective; however, it forms plaques at a multiplicity of infection (moi) greater than one and transduces the tetracycline-resistance determinant to tetracycline-sensitive cells. Analysis of its DNA by restriction endodeoxyribonucleases revealed insertion of the intact transposon Tn10 of 9300 bp in the E fragment, formed during the action of EcoRI, at a distance of 16,800 bp from the pac site.

DNA Transposable Elements↗

PubMed1984

The role of DNA methylation in virus replication: inhibition of frog virus 3 replication by 5-azacytidine.

Frog virus 3 (FV3) DNA is the most highly methylated DNA of any known DNA virus; about 20% of the cytosine residues in FV3 DNA are methylated (D. Willis and A. Granoff, 1980, Virology 107, 250-257). To understand the role of DNA methylation in virus replication, we have examined the effect of 5-azacytidine, a drug that inhibits DNA methylation. 5-Azacytidine (10 microM) reduced the production of infectious FV3 by 100-fold or more and inhibited methylation of viral DNA by about 80%. Inhibition of DNA methylation did not affect viral gene expression since there was no detectable inhibition of virus-specific RNA or protein synthesis in 5-azacytidine-treated cells. In contrast, the size of the replicating DNA measured under completely denaturing conditions, was much smaller than that found during infection in the absence of drug. These results suggest that the undermethylated DNA was susceptible to endodeoxyribonuclease(s). Additionally, electron microscopic examination of FV3-infected, 5-azacytidine-treated cells revealed that preformed capsids remained empty or were only partially filled with viral DNA. Based on these data, it is suggested that methylation of DNA protects it from endonucleolytic cleavage and that the integrity of genomic DNA is required for its proper packaging into virions.

Animals↗

PubMed1982

Bvui: a site-specific endonuclease from Bacillus vulgatis.

A site-specific endodeoxyribonuclease was partially purified from an extract of osmotically shocked spheroplasts of a Bacillus vulgatis strain; the enzyme has been designated BvuI and its activity was characterized. The locations of BvuI-generated cleavages on bacteriophage lambda and M13 derivatives mp7, mp8 and mp9, SV40 and PBR322 DNA molecules were determined. BvuI was shown to recognize the DNA sequence decreases 5'-G-Pu-G-C-Py-C-3' 3'-C-Py-C-G-Pu-G-5' increases and cleaves it at the positions indicated by arrows. Two identical BvuI recognition sites separated by fourteen nucleotide pairs were shown to occur within the tetracycline resistance gene of PBR322; BvuI should be useful for molecular cloning in that plasmid vector.

Bacillus↗

PubMed1994

The apoptosis endonucleases: cleaning up after cell death?

The term apoptosis describes the predictable structural changes associated with many forms of programmed cell death. One of the first visible events of apoptosis is the collapse of the nucleus. Nuclear degradation is manifested by digestion of chromatin into nucleosome-sized fragments or multiples of these. This digestion of DNA is enzymatic, and several attempts have been made to characterize apoptosis-specific endodeoxyribonucleases. Although there are strong candidates for such enzymes, direct evidence for their role in apoptosis is yet to be provided.

Journal Article↗

PubMed1975

Deoxyribonucleic acid polymerases of Euglena gracilis. Primer-template utilization of and enzyme activities associated with the two deoxyribonucleic acid polymerases of high molecular weight.

The two high-molecular-weight DNA polymerases from Euglena gracilis, pol A (mol. wt. 190 000) and pol B (mol. wt. 240 000), were differentiated on the basis of associated enzymic activities and primer-template utilization. Neither enzyme had endodeoxyribonuclease activity, but pol B, like pol B of yeast and the corresponding enzyme from Tetrahymena pyriformis, exhibited at least one other nuclease activity directed against denatured DNA and the RNA of an RNA-DNA hybrid. These nuclease functions preferred an alkaline pH and Mg2+. Pol B also exhibited nucleoside diphosphokinase activity. Both enzymes were active with 'activated' DNA and poly[d(A-T)] as primer-templates and were sensitive, especially pol B, to inhibition by excess of native or heat-denatured DNA. Pol B also utilized oligo[d(T)] and poly(A) templates under certain conditions, whereas pol A exhibited only slight activity with poly[d(A)]. (U)6 was not used as a primer by either enzyme.

Cell-Free System↗

PubMed2000

Site-specific restriction endonucleases in cyanobacteria.

AIM: Planktic cyanobacteria were screened for endodeoxyribonucleases. Principal component analysis (PCA) was employed to demonstrate a potential relationship between certain enzymes and a group of cyanobacteria. The data were obtained from a data bank and this study. METHODS AND RESULTS: Enzymes were partially purified using column chromatography. Anabaena strains contained Asp83/1I (5'-TTCGAA-3'), Asp83/1II (5'-GGCC-3'), Asp90I (5'-ACRYGT-3') and five isoschizomeric enzymes (5'-ATCGAT-3'). Aphanizomenon and Microcystis strains contained ApcTR183I (5'-TGCGCA-3') and Msp199I (5'-CCGG-3'), respectively. Planktothrix strains possessed Psc2I (5'-GAANNNNTTC-3'), Psc27I and Psc28I (5'-TTCGAA-3'). PCA showed that the most common cyanobacterial endonuclease types were AvaII, AvaI and AsuII. CONCLUSIONS: All planktic cyanobacteria studied contained restriction endonucleases. The defined restriction endonucleases were isoschizomers of known enzymes. The Nostoc and the Spirulina genera had an association, while the majority of the genera had no association with certain endonuclease type(s). SIGNIFICANCE AND IMPACT OF THE STUDY: The defined enzymes in this study and the estimated trend in the endonuclease type distribution allow more efficient avoidance of cyanobacterial restriction barriers.

Anabaena↗

PubMed1976

Bacteriophage T5-induced endonucleases that introduce site-specific single-chain interruptions in duplex DNA.

Four site-specific endodeoxyribonucleases have been partially purified from extracts of bacteriophage T5-infected Escherichia coli by gel filtration and affinity chromatography on single- and double-stranded DNA. The enzymes were detected and characterized by agarose gel electrophoresis of alkali-denatured digestion products. None of the four is found in uninfected cells. In the presence of a divalent cation, all four endonucleases make ligase-repairable, single-chain interruptions at specific sites in the duplex DNA of several bacteriophages (lambda, T7, and T5) and a mammalian virus (adenovirus 2). These activities are not stimulated by ATP. None of the four is active on single-stranded DNA. The fragments produced by each enzyme from ligase-repaired T5 DNA do not correspond to those derived from mature T5 DNA. Each of the enzymes is able to cleave the intact strand of T5 DNA.

Coliphages↗

PubMed1977

Cloning of the silk fibroin gene and its flanking sequences.

Thirteen Escherichia coli clones containing the whole or a part of the fibroin gene (16 kilobases long) have been isolated. The starting material was DNA extracted from the posterior silk glands of Bombyx mori. Most clones were obtained from sheared DNA fragments linked by poly(dA)-poly(dT) joints to the plasmid pMB9. One of them includes the 5' end of the fibroin gene with a flanking sequence of 12 kilobases, and another includes the 3' end of the gene with a flanking sequence of about 1 kilobase. One clone was obtained by ligation, to pMB9, of a fragment generated by endodeoxy-ribonuclease EcoRI. This clone has a 21-kilobase insertion that probably includes the entire fibroin gene with flanking sequences at both ends. The cleavage sites for endodeoxyribonucleases EcoRI, HindIII, and BamHI have been established for the cloned sequences.

Base Sequence↗

PubMed1982

Involvement of outside DNA sequences in the major kinetic path by which EcoRI endonuclease locates and leaves its recognition sequence.

We have examined the kinetics of the interaction between endodeoxyribonuclease EcoRI (EC 3.1.23.13) and nine linear DNA fragments that range in size between 34 and 6,200 base pairs and contain the EcoRI site of plasmid pBR322 in a central location. The kinetic parameters governing both formation and decay of specific endonuclease . DNA complexes increase 8-fold with increasing chain length over this size range. In contrast, equilibrium competition experiments demonstrated that the intrinsic affinity of endonuclease for its recognition sequence is independent of DNA chain length over this range. Thus, DNA sequences outside the recognition site enhance the rate at which EcoRI endonuclease locates or leaves its recognition site without affecting the intrinsic thermodynamic parameters of site-specific interaction. These results are consistent with a facilitated diffusion mechanism for specific DNA site location by this enzyme.

Base Sequence↗

PubMed1972

The form and structure of kinetoplast DNA of Crithidia.

Cesium chloride centrifugation of each of the DNAs extracted from eight strains of Crithidia revealed a main band at rho = 1.717 g/cm(3) and a satellite band varying from rho = 1.701 to 1.705 g/cm(3) for the different strains By electron microscopy each DNA was shown to include circular molecules, 0.69-0.80 micro in mean contour length, and large, topologically two-dimensional masses of DNA in which the molecules appeared in the form of rosettes. DNA isolated from kinetoplast fractions of Crithidia acanthocephali was shown to consist of light satellite DNA and to be mainly in the form of large masses, 0.8 micro (mol wt = 1.54 x 10(6) daltons) circular molecules, and a few long, linear molecules. The results of experiments involving ultracentrifugation, heating, and quenching, sonication, and endodeoxyribonuclease digestion, combined with electron microscopy, are consistent with the following hypothesis. The large DNA masses are associations of 0.8 micro circles which are mainly covalently closed. The circles are held together in groups (the rosettes) of up to 46 by the topological interlocking of each circle with many other circles in the group. A group of circles is attached to an adjacent group by one or more circles, each interlocking with many circles of both groups. Each of the associations comprises, on the average, about 27,000 circles (total mol wt approximately 41 x 10(9) daltons). A model is proposed for the in situ arrangement of the associations which takes into consideration their form and structure, and appearance in thin sections

Acridines↗

PubMed1978

HeLa DNA polymerase alpha activity in vitro: specific stimulation by a non-enzymic protein factor.

A non-enzymic protein factor that increases the in vitro rate of synthesis by HeLa DNA polymerase alpha 15- to 30-fold with denatured DNA as template has been partially purified from the cytoplasmic fraction of HeLa cells. The stimulatory effect is highly specific for HeLa DNA polymerase alpha and for DNA templates that contain extensive regions of single-strandedness. Synthesis with denatured DNA as template presumably proceeds from 3'-hydroxyl termini formed at loop-back regions since the synthesized DNA product and template are covalently linked. The stimulatory protein factor chromatographs as a basic protein, has an approximate molecular weight of 30,000 daltons and binds with moderate affinity to denatured DNA cellulose, being eluted by o.4M NaCl. The purified factor lacks detectable DNA polymerase, exo- and endodeoxyribonuclease and RNA polymerase activities. It also does not promote helix-coil transitions with poly[d(A-T)] and Clostridium perfringens DNA.

DNA Polymerase II↗

PubMed1984

Purification and characterization of a factor stimulating DNA polymerase alpha activity from mouse FM3A cells.

A protein factor which stimulates DNA polymerase alpha activity on heat-denatured DNA has been purified from mouse FM3A cells. The final preparation had a specific activity of 43,000 units/mg protein and lacked detectable DNA polymerase, RNA polymerase, DNA-dependent- and independent ATPase, exo- and endodeoxyribonuclease and phosphatase activities. The stimulating factor sedimented at 2.9S in a glycerol gradient. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the glycerol gradient fraction revealed the presence of a major band of 36,000 daltons, the amount of which corresponded well with the level of stimulating activity. The stimulation by the factor was specific for heat-denatured DNA, and a little or no stimulation was observed with native DNA, ribo- and deoxyribohomopolymers and single stranded circular DNA. Alkaline sucrose gradient sedimentation analysis of the reaction products revealed that newly synthesized DNA was covalently linked to the termini of heat-denatured DNA. The average chain length of the elongated span determined by the digestion with micrococcal nuclease and phosphodiesterase II, did not differ between in the presence and absence of the stimulating factor, suggesting that the stimulation by the factor was due to the increase in the initiation frequency of DNA synthesis from the 3'-hydroxyl terminus of heat-denatured DNA.

Animals↗

PubMed1981

DNA-binding proteins in frog virus 3-infected cells.

At least 12 virus-induced DNA-binding proteins with mol. wt. ranging from 14 X 10(3) have been isolated from frog virus (FV 3)-infected fathead minnow cells by DNA affinity chromatography. Two enzymic activities, DNA-dependent DNA polymerase and endodeoxyribonuclease, were present in the DNA-binding proteins; these enzymic activities were similar to those induced by FV 3 in infected cells. A single species of DNA-binding proteins with a mol. wt of 36 000 had very high affinity for single-stranded DNA, but a low one for double-stranded DNA. Proteins with such characteristic affinity for single-stranded DNA destabilize the DNA helix and are essential for viral DNA replication. Thus, the 36 000 mol. wt. DNA-binding protein is a candidate for such a role in FV 3 DNA replication.

Animals↗

PubMed1980

Studies on the mechanism of transcription of nucleosomal complexes.

The mechanism of transcription by Escherichia coli RNA polymerase holoenzyme of chromatin assembled in vitro has been studied by two approaches. Using digestion with endodeoxyribonuclease EcoRI as a probe of mobility, it was found that nucleosome movement is slow compared to the time taken for RNA polymerase to transcribe through regions organised into nucleosomes. However, transcription leads to at least some displacement of nucleosomes relative to their original site on the DNA. In the second approach chromatin was reconstituted from extensively crosslinked histone octamers and simian virus 40 DNA. RNA chain elongation on this template is inhibited relative to non-crosslinked chromatin. This can be related to a decrease in the ability of the cross-linked histone octamers to dissociate from the DNA.

Chromatin↗

PubMed1982

DNA-processing activities associated with the purified alpha, beta 2, and alpha beta molecular forms of avian sarcoma virus RNA-dependent DNA polymerase.

The RNA-dependent DNA polymerase purified from B77 avian sarcoma virus exhibited two distinct DNA-processing activities. The alpha and beta 2 isoenzymes possessed an endodeoxyribonuclease activity capable of nicking simian virus 40 superhelical DNA, whereas the alpha beta isoenzyme performed as an untwisting topoisomerase. Both activities associated with the three molecular forms of the retroviral DNA polymerase were dependent on the presence of either Mn2+ or Mg2+ ions. From analysis of the denaturated DNA products, it is apparent that the alpha and beta 2 isoenzymes introduced two nicks, one per each strand in the superhelical simian virus 40 DNA molecules, whereas the alpha beta polymerase converted these supercoiled molecules to the relaxed covalently closed circular form. The notion that the DNA-processing activities are located on the DNA polymerase molecules was supported by the following: (i) the three isoenzymes were of a high purity; (ii) the activities cosedimented in glycerol gradients with the DNA polymerase activities of the alpha, beta 2, and alpha beta molecular forms; and (iii) immunoglobulin directed against the purified polymerase immunoprecipitated the DNA-processing activities. Chemical treatments of the DNA polymerase molecules (with pyridoxalphosphate, iodoacetamide, and sulfhydryl reagents), which inhibited the polymerase activity, also suppressed the endonucleolytic and topoisomerase activities, suggesting that cystein and amino groups play an important role in the active sites of the DNA-processing activities as well.

Alpharetrovirus↗

PubMed1992

Genome digestion is a dispensable consequence of physiological cell death mediated by cytotoxic T lymphocytes.

We examined virally transformed murine fibroblast clones as targets for cytotoxic T lymphocyte (CTL)-triggered lysis and genome digestion. Strikingly, while all clones were essentially equivalent in the ability to be lysed, one clone, SV3T3-B2.1, failed to exhibit genome digestion associated with CTL attack. Other aspects of the physiological cell death process, including loss of adhesion and nuclear envelope breakdown (lamin phosphorylation and solubilization), were not altered in this clone. The absence of genome digestion associated with CTL-induced cell death correlated with the absence of endodeoxyribonuclease activity in the nuclei of that clone. Characterization of the activity affected identifies a calcium-dependent, DNase I-like endonuclease of approximately 40 kDa, normally present constitutively in all cell nuclei, as the enzyme responsible for genome digestion associated with CTL-mediated cell death. These observations indicate that neither genome digestion per se nor its consequences [such as activation of poly(ADP-ribose) polymerase] are essential for cell death resulting from the triggering of this cell suicide process.

Animals↗

PubMed1986

An immunochemical study of Neurospora nucleases.

Nucleases derived from Neurospora crassa mycelia with neutral single-strand (ss) endodeoxyribonuclease activity have been examined by immunochemical techniques and by sodium dodecyl sulfate - DNA gel electrophoresis. All of the intracellular nucleases, which have different divalent metal ion requirements, different strand specificities with single- and double-strand DNA, different modes of action on DNA and RNA, and other distinguishing characteristics, are immunochemically related to Neurospora endo-exonuclease. The evidence indicates that these enzymes are derived from one or more related large, inactive (precursor?) polypeptides that are first converted to 75- to 80-kdalton active polypeptide(s) which are very protease sensitive. Further limited proteolysis results in the production of the various active forms of nuclease studied here. Some proteolytic conversions may occur in a controlled manner in vivo in different cell compartments, but others are very likely artifacts resulting from uncontrolled proteolysis during extraction and isolation. The intracellular forms of Neurospora endo-exonuclease are immunologically cross-active with ss-DNA-binding nucleases isolated from Aspergillus nidulans and Saccharomyces cerevisiae. They are not immunochemically related to two extracellular Neurospora nucleases, the pancreatic DNase-I-like DNase A and a ss-specific exonuclease, and they are also not related to other fungal and plant nucleases with ss-specific endonuclease activity such as the S1 nuclease of Aspergillus oryzae, the P1 nuclease of Penicillium citrinum, and mung bean nuclease.

Antibodies↗

PubMed1977

Endonuclease V of Escherichia coli.

A small endodeoxyribonuclease )2.3 S) that is active on single-stranded DNA has been extensively purified from Escherichia coli so as to be free of other known DNases. It has an alkaline pH optimum (9.5), requires Mg2+, and makes 3'-hydroxy and 5'-phosphate termini. The nuclease nicks duplex DNA, particularly if treated with OsO4, irradiated with ultraviolet light, or exposed to pH 5. The uracil-containing duplex DNA from the Bacillus subtilis phage PBS-2 is an especially good substrate; it is made acid-soluble by levels of the enzyme which fail to produce any acid-soluble material in other single-stranded or duplex DNAs. Neither RNA nor RNA-DNA hybrid are degraded by the enzyme. The enzyme specificity suggests that it might act at abnormal regions in DNA, so that its in vivo function could be to initiate an excision repair sequence. Its high activity on uracil-containing DNA could imply that the enzyme provides an alternative mechanism for excising uracil residues from DNA to the pathway utilizing uracil-DNA N-glycosidase. We suggest that this enzyme be designated as endonuclease V of E. coli.

Cations, Divalent↗