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Results for “Exonucleases”

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At least 847 records · Page 47Linked to original sources
PubMed1977

A rapid method for preparation of calf spleen exonuclease.

Chromatography on Concanavalin A sepharose is a simple procedure for partial purification of spleen phosphodiesterase. This procedure removes much, but not all, of the ribonuclease activity found as contaminant in most spleen phosphdisterase preparations.

Animals↗
PubMed1981

Detection of the catalytic activities of DNA polymerases and their associated exonucleases following SDS-polyacrylamide gel electrophoresis.

A method is described to detect DNA polymerases and nucleases in homogeneous or crude enzyme preparations after electrophoresis in SDS-polyacrylamide gels(2) containing the appropriate template or substrate. DNA polymerases are electrophoresed in a gel containing gapped calf thymus DNA and after a renaturation treatment, the gel is incubated in a reaction mixture in which one deoxyribonucleoside triphosphate is [alpha-32P]-labelled. Incorporation of radioactivity into DNA is detected at the vicinity of the polymerase band by autoradiography. An associated nuclease activity can be measured after electrophoresis in a gel containing 32P-labelled gapped DNA, when nucleolytic digestion is seen as a clear band in the resulting autoradiogram. The gels can subsequently be stained with Coomassie blue to establish identical molecular weights of polymerase, nuclease and protein bands. Applications of this technique are discussed.

Bacillus subtilis↗
PubMed1999

Reconstitution of a minimal RNA degradosome demonstrates functional coordination between a 3' exonuclease and a DEAD-box RNA helicase.

The RNA degradosome is a multiprotein complex required for the degradation of highly structured RNAs. We have developed a method for reconstituting a minimal degradosome from purified proteins. Our results demonstrate that a degradosome-like complex containing RNase E, PNPase, and RhlB can form spontaneously in vitro in the absence of all other cellular components. Moreover, ATP-dependent degradation of the malEF REP RNA by the reconstituted, minimal degradosome is indistinguishable from that of degradosomes isolated from whole cells. The Rne protein serves as an essential scaffold in the reconstitution process; however, RNase E activity is not required. Rather, Rne coordinates the activation of RhlB dependent on a 3' single-stranded extension on RNA substrates. A model for degradosome-mediated degradation of structured RNA is presented with its implications for mRNA decay in Escherichia coli.

ATP-Binding Cassette Transporters↗
PubMed1978

Yeast DNA polymerases: antigenic relationship, use of RNA primer and associated exonuclease activity.

Highly purified preparation of DNA polymerases A and B from yeast were compared with respect to antigenic relationship, ability to use ribonucleotide primers and associated nuclease activity. The following results were obtained. 1. Antiserum directed against DNA polymerase A inhibits this enzyme but does not interfere with activity of DNA polymerase B or of mitochondrial DNA polymerase, nor does it precipitate the latter two enzymes. 2. DNA polymerase A is capable of using oligo(ribouridylic acid) as a primer for the polymerization of dTMP. This reaction is not catalyzed by polymerase B to any significant extent. 3. Whereas DNA polymerase A is devoid of nuclease activity, DNA polymerase B catalyses an exonucleolytic release of mononucleotide units from the 3' end of polynucleotides. The results of several experiments suggest that this nuclease activity is associated with the DNA polymerase B molecule.

DNA-Directed DNA Polymerase↗
PubMed2004

Design of a 5' exonuclease-based real-time PCR assay for simultaneous detection of Bacillus licheniformis, members of the 'B. cereus group' and B. fumarioli in gelatine.

AIMS: The design of a fast, sensitive and specific detection method for Bacillus licheniformis, members of the 'B. cereus group' and B. fumarioli in gelatine. METHODS AND RESULTS: Specific Taqman probes were designed and tested in a real-time PCR setting. A specific fluorescent signal could be obtained for all gelatine isolates attributed to these species in one single real-time PCR reaction. After sample preparation, a gelatine sample spiked with 1 CFU provided enough template DNA for a significant signal. CONCLUSION: The potential of a real-time PCR assay for simultaneous detection of B. licheniformis, members of the 'B. cereus group' and B. fumarioli in gelatine is demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: Implementation of the assay in gelatine producing plants may shorten delivery terms and inform on hazards to public health and suitable remediation procedures.

Bacillus↗
PubMed1982

Exonuclease activity from Pseudomonas aeruginosa which is missing in phenotypically restrictionless mutants.

A phenotypically restrictionless strain of Pseudomonas aeruginosa was found to lack a deoxyribonuclease specific for linear duplex DNA. The purified enzyme had an optimum pH of 8.5, required MgCl2 (10 mM) for maximum activity, and did not require ATP. Neither the degradation of heat-denatured DNA nor the degradation of bacteriophage F116 DNA was detected. The genome of bacteriophage F116 was shown to possess single-stranded terminal regions, which account for the resistance to degradation and for the ability of the phage to transfect restriction-proficient strains.

Bacteriophages↗
PubMed1984

Restoration of viability to an Escherichia coli mutant deficient in the 5'----3' exonuclease of DNA polymerase I.

An Escherichia coli polA (Ex) mutant that is usually inviable at restrictive temperatures (43 degrees C) was found to grow normally at 43 degrees C when incubated in the presence of a membrane-containing fraction prepared from E. coli. This membrane fraction causes anaerobic conditions that are necessary but not sufficient for restoration of viability since some component present in the membrane fraction is also required for colony formation at 43 degrees C. The accumulation of small DNA fragments typical of aerobic growth of the polA(Ex) mutant was also seen under anaerobic conditions. The polA(Ex) strain was also much more sensitive than the isogenic wild-type strain to hydrogen peroxide.

Anaerobiosis↗
PubMed1998

Oligoribonuclease is encoded by a highly conserved gene in the 3'-5' exonuclease superfamily.

Oligoribonuclease, a 3'-to-5' exoribonuclease specific for small oligoribonucleotides, was purified to homogeneity from extracts of Escherichia coli. The purified protein is an alpha2 dimer of 40 kDa. NH2-terminal sequence analysis of the protein identified the gene encoding oligoribonuclease as yjeR (o204a), a previously reported open reading frame located at 94 min on the E. coli chromosome. However, as a consequence of the sequence information, the translation start site of this open reading frame has been revised. Cloning of yjeR led to overexpression of oligoribonuclease activity, and interruption of the cloned gene with a kanamycin resistance cassette eliminated the overexpression. On the basis of these data, we propose that yjeR be renamed orn. Orthologs of oligoribonuclease are present in a wide range of organisms, extending up to humans.

Bacterial Proteins↗
PubMed2026

A structural bridge between dengue virus tandem xrRNAs facilitates coordination of exonuclease resistance.

Orthoflavivirus RNA genomes resist host 5'-3' exoribonucleases to produce subgenomic flaviviral RNAs (sfRNAs). This resistance is conferred by exoribonuclease-resistant RNA (xrRNA) structures within the viral 3' untranslated region that often occur in tandem, and whose function can be coupled. In dengue virus serotype 2 (DENV2), this coupling results in changing patterns of sfRNA identity and abundance associated with the ability of the virus to adapt to host vs. vector infections. The physical basis of this coupling was unknown. Using a combination of virology, biochemistry, bioinformatics, structural biology, and biophysics, we explored the structural and sequence determinants of tandem xrRNA coupling in DENV2. We discovered that the spatial proximity, order, and structural integrity of the tandem xrRNAs are all important for coupling. Furthermore, an unpaired A-rich linker that lies between the two xrRNAs is essential in stabilizing a specific structure that correlates to coupling. This A-rich sequence likely forms tertiary contacts with an adjacent stem-loop structure to form a physical bridge between the two xrRNAs, a finding that is supported by a mid-resolution cryo-electron microscopy (cryo-EM) map of the DENV2 tandem xrRNAs. Disruption of the structure of this bridge by mutation changes the relative orientation or spacing between the tandem xrRNAs, which is correlated to their functional coupling. These findings help provide an explanation for the coupling between tandem xrRNAs, suggesting a new mechanistic hypothesis in which the two tandem xrRNAs can simultaneously encounter Xrn1.IMPORTANCEDengue virus (DENV) generates non-coding subgenomic flaviviral RNAs (sfRNAs) that affect several cellular pathways and are important for successful infection. These sfRNAs are formed by structured RNA elements in the viral genome called exoribonuclease-resistant RNAs (xrRNAs), which fold into a distinct three-dimensional topology to block degradation by host cell exoribonucleases and often occur in tandem. Specific patterns of sfRNAs made during infection are important for host vs. vector fitness, and in DENV2, this pattern depends on functional coupling between tandem xrRNAs. However, the source of this functional coupling was unknown. We determined that an unpaired A-rich linker between the tandem xrRNAs is necessary for creating a structural bridge between the tandem xrRNAs. This bridge appears to favor a specific orientation between the tandem xrRNAs that is correlated to coupling and therefore to the patterns and relative abundance of sfRNAs produced during infection.

Dengue Virus↗
PubMed2001

Polynucleotide phosphorylase functions as both an exonuclease and a poly(A) polymerase in spinach chloroplasts.

The molecular mechanism of mRNA degradation in the chloroplast consists of sequential events including endonucleolytic cleavage, the addition of poly(A)-rich sequences to the endonucleolytic cleavage products, and exonucleolytic degradation by polynucleotide phosphorylase (PNPase). In Escherichia coli, polyadenylation is performed mainly by poly(A)-polymerase (PAP) I or by PNPase in its absence. While trying to purify the chloroplast PAP by following in vitro polyadenylation activity, it was found to copurify with PNPase and indeed could not be separated from it. Purified PNPase was able to polyadenylate RNA molecules with an activity similar to that of lysed chloroplasts. Both activities use ADP much more effectively than ATP and are inhibited by stem-loop structures. The activity of PNPase was directed to RNA degradation or polymerization by manipulating physiologically relevant concentrations of P(i) and ADP. As expected of a phosphorylase, P(i) enhanced degradation, whereas ADP inhibited degradation and enhanced polymerization. In addition, searching the complete Arabidopsis genome revealed several putative PAPs, none of which were preceded by a typical chloroplast transit peptide. These results suggest that there is no enzyme similar to E. coli PAP I in spinach chloroplasts and that polyadenylation and exonucleolytic degradation of RNA in spinach chloroplasts are performed by one enzyme, PNPase.

Chloroplasts↗