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At least 613 records · Page 34Linked to original sources
PubMed1994

Endo-exonucleases: enzymes involved in DNA repair and cell death?

Endo-exonucleases from E. coli to man, although very different proteins, are multifunctional enzymes with similar enzymatic activities. They probably have two common but opposing biological roles. On the one hand, they promote survival of the organism by acting in recombination and recombinational DNA repair to diversify and help preserve the genome intact. On the other hand, they degrade the genomic DNA when it is damaged beyond repair. This ensures elimination of heavily mutagenized cells from the population.

Animals↗
PubMed2006

Minimizing loss of sequence information in SAGE ditags by modulating the temperature dependent 3' --> 5' exonuclease activity of DNA polymerases on 3'-terminal isoheptyl amino groups.

Numerous steps are required to prepare a sequencing library for serial analysis of gene expression (or SAGE) from an original mRNA sample. The presence of inefficiencies, however, can lead to a cumulative loss of sample during processing which can yield a library of short sequence tags (SSTs) that represents only a minute fraction of the original starting sample, potentially compromising the quality of the analysis and necessitating relatively large amounts of starting material. We show here that commonly observed higher molecular weight (HMW) amplification products observed following the PCR amplification of ditags are a direct result of the presence of HMW ligation products created during ditag formation. Using model tags, we demonstrate that the formation of these HMW ligation products becomes permissible following the release of the 3'-terminal isoheptyl amine (3'-IHA) from the SST during the fill-in reaction with the Klenow fragment (KF) of DNA polymerase (DNAP) I and is mediated by its 3' --> 5' exonuclease activity. We further show that the incorporation of SSTs into HMW ligation products can lead to a loss of sequence information from SAGE analysis, potentially skewing sequencing results away from the true distribution in the original sample. By modifying fill-in conditions through the use of Vent DNAP at 12 degrees C and by including terminal phosphorothioate linkages within the SAGE adaptors to specifically inhibit exonucleolytic removal of the 3'-terminal amine, we are able to maximize the yield of ditags and bypass the need for gel purification via PAGE following PCR. The modifications described here, combined with the modifications described previously by our group for adaptor ligation, ensure that the full sequence information content in SSTs derived from the transcriptome is preserved in the pool of amplified ditags prior to the creation of a SAGE library.

3' Untranslated Regions↗
PubMed2006

The exonuclease ISG20 mainly localizes in the nucleolus and the Cajal (Coiled) bodies and is associated with nuclear SMN protein-containing complexes.

We have previously shown that ISG20, an interferon (IFN)-induced gene, encodes a 3' to 5' exoribonuclease member of the DEDD superfamily of exonucleases. ISG20 specifically degrades single-stranded RNA. In this report, using immunofluorescence analysis, we demonstrate that in addition to a diffuse cytoplasmic and nucleoplasmic localization, the endogenous ISG20 protein was present in the nucleus both in the nucleolus and in the Cajal bodies (CBs). In addition, we show that the ectopic expression of the CBs signature protein, coilin, fused to the red fluorescent protein (coilin-dsRed) increased the number of nuclear dots containing both ISG20 and coilin-dsRed. Using electron microcopy analysis, ISG20 appeared principally concentrated in the dense fibrillar component of the nucleolus, the major site for rRNA processing. We also present evidences that ISG20 was associated with survival of motor neuron (SMN)-containing macromolecular nuclear complexes required for the biogenesis of various small nuclear ribonucleoproteins. Finally, we demonstrate that ISG20 was associated with U1 and U2 snRNAs, and U3 snoRNA. The accumulation of ISG20 in the CBs after IFN treatment strongly suggests its involvement in a new route for IFN-mediated inhibition of protein synthesis by modulating snRNA and rRNA maturation.

Cell Nucleolus↗
PubMed1999

Processive proofreading and the spatial relationship between polymerase and exonuclease active sites of bacteriophage phi29 DNA polymerase.

phi29 DNA polymerase is a multifunctional enzyme, able to incorporate and to proofread misinserted nucleotides, maintaining a very high replication fidelity. Since both activities are functionally separated, a mechanism is needed to guarantee proper coordination between synthesis and degradation, implying movement of the DNA primer terminus between polymerization and 3'-5' exonuclease active sites. Using single-turnover conditions, we have demonstrated that phi29 DNA polymerase edits the polymerization errors using an intramolecular pathway; that is, the primer terminus travels from one active site to the other without dissociation from the DNA. On the other hand, by using chemical tags, we could infer a difference in length of only one nucleotide to contact the primer strand when it is in the polymerization mode versus the editing mode. Using the same approach, it was estimated that phi29 DNA polymerase covers a DNA region of ten nucleotides, as has been measured in other polymerases using different techniques.

Bacillus Phages↗
PubMed2025

Transcriptome-Wide Analysis of the 5' Cap Status of RNA Using 5' Monophosphate-Dependent Exonuclease Digestion and RNA Sequencing.

Eukaryotic mRNAs carry an N7-methylguanosine (m7G) cap structure at their 5' extremity, which protects them from the degradation by 5'-3' exoribonucleases and plays a pivotal role in mRNA metabolism, promoting splicing, nuclear export, and translation. Decapping, the enzymatic process that removes this structure, is a key event during cytoplasmic mRNA 5'-3' decay, leading to the degradation of the transcript body by Xrn1. In this chapter, we describe a procedure to assess the cap status of RNA at the transcriptome level. It is based on a treatment of total RNA extracts with a 5' monophosphate-dependent exonuclease, which like Xrn1 specifically degrades decapped RNAs harboring 5' monophosphate extremities, but not RNAs with intact m7G cap. The digested RNAs are then analyzed by RNA sequencing.

Exoribonucleases↗
PubMed1975

Involvement of Escherichia coli DNA polymerase-I-associated 5' in equilibrium 3' exonuclease in excision-repair of UV-damaged DNA.

From comparative studies between Escherichia coli PolA107 cells (lacking 5' in equilibrium 3' exonucleoytic activity associated with DNA polymerase I) and the isogenic wild-type strain, and between the purified DNA polymerase I preparations isolated from these strains, it can be concluded that the 5' in equilibrium 5' exonuclease is involved in excision of pyrimidine dimers in E. coli. Evidence is presented that the polA107 mutation is located on that part of the DNA polymerase I gene coding for the small fragment on which 5' in equilibrium 3' exonucleolytic activity is found.

DNA↗
PubMed1975

DNA degradation in minicells of Escherichia coli K-12. II. Effect of recA1 and recB21 mutations on DNA degradation in minicells and detection of exonuclease V activity.

The properties of minicell producing mutants of Escherichia coli deficient in gentic recombination were examined. Experiments were designed to test recombinant formation in conjugal crosses, survival following UV-irradiation in cells, and the state of DNA metabolism in minicells. The REC- phenotypes are unaffected by min+/- genotypes in whole cells. In contrast to minicells produced by rec+ parental cells, minicells from a recB21 strain have limited capacity to degrade linear, Hfr transfereed DNA. The lack of a functional recA gene product, presumably involved in inhibiting the recBC nuclease action(s), permits unrestricted Hfr DNA breakdown in minicells produced by a recA1 strain. This results in an increase in TCA soluble products and in the formation of small DNA molecules that sediment near the top of an alkaline sucrose gradient. Unlike the linear DNA, circular duplex DNA from plasmids R 64-11 or lambdadv, segregated into the minicells, is resistant to breakdown. By using in vitro criteria, and [32P]-labelled linear DNA from bacteriophage T7 for substrate, we found that the ATP-dependent exonuclease of the recBC complex (exo V) is present in rec+ and recA- minicells, and is lacking in the recB21 mutant. In fact, the absence of a functional exo V in recBC- minicells results in isolation of larger than average Hfr DNA from minicells. We suggest that recombination (REC) enzymes segregate into the polar minicells at the time of minicell biogenesis. This system should be useful for studies on DNA metabolism and functions of the recBC and recA gene products.

DNA, Bacterial↗
PubMed1981

The requirement for both DNA polymerase and 5' to 3' exonuclease activities of DNA polymerase I during Tn5 transposition.

By assaying transposition of Tn5 from lambda b221 cI857 rex::Tn5 (Berg 1977) in polA-proficient and deficient cells, both the polymerase activity and 5' to 3' exonuclease activity of DNA polymerase I have been shown to be required for transposition. This requirement could not be observed in three other systems in which the transposon donor replicon had existed in the PolA-proficient and deficient cells before the transposition event to be assayed occurred. By analogy to Tn3, this may indicate that the repressor encoded by Tn5 has already been expressed and hence become rate-limiting in the overall transposition process, even PolA-deficient cells still possessing a residual activity. One PolA mutant was found among more than 50 transposition deficient (tnp) mutants isolated by the use of lambda b221 cI857 rex::Tn5.

DNA Polymerase I↗
PubMed2003

Increased sensitivity of B-cell clonality analysis in formalin-fixed and paraffin-embedded B-cell lymphoma samples using an enzyme blend with both 5'-->3' DNA polymerase and 3'-->5' exonuclease activity.

Polymerase chain reaction (PCR)-based detection of immunoglobulin heavy chain (IgH) gene rearrangement for determination of B-cell clonality needs to be simple but optimally sensitive. Efficient IgH PCR analysis can be hampered by sequence variability in the template DNA, despite of the use of degenerative primers. To improve sensitivity of the B-cell clonality analysis in formalin-fixed and paraffin-embedded (FFPE) tissues, we have performed framework three-area (FR3)/joining gene (JH) IgH PCR utilizing an enzyme blend (r Tth DNA Polymerase, XL) providing both 5'-->3' polymerase and 3'-->5' exonuclease activities. The DNA samples were extracted from FFPE biopsies of 43 mature B-cell lymphoma cases of so-called germinal center and post-germinal center origin, including 6 nodal follicular lymphomas (FL), 15 gastric mucosa-associated lymphoid tissue (MALT) lymphomas, and 22 gastric diffuse large B-cell lymphomas (DLBCL). Of the cases, 31 (17 DLBCL and 14 MALT lymphoma) represented small endoscopic biopsies. Serial dilutions of target DNA were applied to avoid inconsistent bands that may be seen when the input amount of template is too low, which can be the case when DNA is extracted from FFPE endoscopic gastric biopsies. Using conventional Taq polymerase, consistent monoclonal product was found in 53% (23/43) of the cases (FL: 67%; MALT lymphoma: 47%; DLBCL: 55%). The r Tth polymerase showed reproducible monoclonal pattern in 72% (31/43) of the cases (FL: 67%; MALT lymphoma: 73%; DLBCL: 73%); the sensitivity is compatible with one that can be detected with conventional FR3/JH PCR in fresh/frozen tissues. In conclusion, the r Tth DNA polymerase greatly improves sensitivity of FR3/JH PCR in FFPE biopsies of mature B-cell lymphomas, most probably by increasing the primer matches during PCR amplification.

DNA Primers↗
PubMed1981

ATP-dependent exonuclease V from Micrococcus luteus. The enzyme-DNA complex, the processive mechanism, and the role of ATP.

Some kinetic predictions of the proposed processive mechanism for the hydrolysis of DNA by the ATP-dependent enzyme exonuclease V have been checked. The method is to trap enzyme molecules not attached to radioactive DNA substrate with an excess of nonradioactive DNA, so that enzyme molecules attached to the radioactive substrate contribute to the liberation of radioactive products only until they dissociate from it. The experiments show that enzyme molecules remain attached to a T7 double-stranded DNA molecule, while hydrolysing it, for about 2 min under our conditions, in agreement with the predictions of the processive mechanism. However, the mechanism of degradation of single-stranded DNA is not processive. Formation of an enzyme-DNA complex is largely dependent on the presence of ATP. This formation does not appear to be synchronous. ATP analogs do not stimulate formation of, nor stabilize, the enzyme-DNA complex. EDTA causes dissociation of enzyme molecules from the DNA complex.

Adenosine Triphosphate↗
PubMed1980

Micrococcus radiodurans surface exonuclease. Dimer to monomer conversion by ionizing radiation-generated aqueous free radicals.

Micrococcus radiodurans possesses an exonuclease firmly bound to a middle cell wall membrane layer. Aqueous OH. radicals generated chemically or by ionizing radiation cause the immediate release of this enzyme into the surrounding medium. The enzyme is located in a hydrophobic site and can also be released by aqueous n-butanol. When extracted by this solvent it is a non-covalently linker dimer and has a molecular weight of 260 000 as determined by gel filtration. When released by radiation generated OH. radicals, the enzyme initially appears in solution as the dimer but is rapidly split by further aqueous radical attack into two 130 000 molecular weight subunits. Hydroxyl radicals are most effective but reducing radicals are also able to monomerize the enzyme. Only the released dimer enzyme is subject to free radical monomerization. Bound dimer enzyme is not split prior to release. No detectable loss of activity or change in catalytic properties accompanies the free radical cleavage of the enzyme. Both subunits of the dimer enzyme possess a tightly bound metal ion (probably Ca2+) required for activity. The monomer but not the dimer enzyme will bind to an anion exchanger. The monomer is susceptible to loss of its metal ion, and consequent inactivation, when exposed to the exchanger in the absence of Ca2+. Besides providing information on some of the immediate non-lethal effects of ionizing radiation, the behavior of this enzyme system demonstrates a potential cellular mechanism by which internally or externally generated free radicals could be utilized by the cell to control various enzymic reactions.

Butanols↗
PubMed1992

Action of Escherichia coli and human 5'----3' exonuclease functions at incised apurinic/apyrimidinic sites in DNA.

The 5'----3' exonuclease activity of E. coli DNA polymerase I and a related enzyme activity in mammalian cell nuclei, DNase IV, are unable to catalyse the excision of free deoxyribose-phosphate from apurinic/apyrimidinic (AP) sites incised by an AP endonuclease. Instead, the sugar phosphate residue is slowly released as part of a short oligonucleotide. These products have been characterised as dimers and trimers by comparison of their retention time on reverse-phase HPLC with reference compounds prepared by acid depurination of a dinucleotide, trinucleotide and tetranucleotide containing a 5'-terminal dAMP residue. The similar mode of action of these enzymes at 5'-incised AP sites provides an explanation for the minority of repair patches larger than one nucleotide observed when AP sites are repaired by E. coli and mammalian cell extracts in vitro and strengthens the functional analogy between the two activities.

Apurinic Acid↗
PubMed1992

Role of exonuclease in the specificity of bacteriophage T7 DNA packaging.

During morphogenesis in vivo, bacteriophage T7 packages and cuts to mature size an end-to-end concatemer of its nonpermuted, terminally repetitious, double-stranded, mature DNA. Efficient production (90-100%) and packaging (20-35%) of concatemers has also been demonstrated in extracts of T7-infected cells (in vitro) (Son, M., Hayes, S. J., and Serwer, P. [1988] Virology 162, 38-46). By use of both this procedure of in vitro DNA packaging and in-gel hybridization to packaged DNA fractionated by agarose gel electrophoresis, the specificity of packaging in vitro is found to depend on the presence of T7 gene 6 exonuclease (p6). In the absence of p6 in vitro, no concatemerization is detected and packaging of DNA nonhomologous to T7 DNA (bacteriophage P22 DNA) is as efficient (0.05-1.1%) as the packaging of monomeric T7 DNA. Addition of p6 in vitro both stimulates the concatemerization-packaging of T7 DNA and suppresses the packaging of P22 DNA. The packaging efficiency for concatemeric T7 DNA is 29-611 x higher than that for monomeric T7 DNA. Inhibition of the packaging of P22 DNA by p6 is correlated with the formation of single-stranded P22 DNA ends. These data are explained by the hypothesis that a DNA molecule with a single-stranded end is packaged less efficiently than the same DNA without the single-stranded end. Testing this hypothesis in vivo reveals that both p6 and gene 3 endonuclease contribute to suppressing the packaging of host DNA.

Capsid↗
PubMed1978

Nucleosomal DNA is digested to repeats of 10 bases by exonuclease III.

Nucleosomes were treated with increasing concentrations of exonuclease III (Exo III) from E. coli. At low levels of Exo III, the heterogeneous distribution of monomers (with associated DNA fragments ranging in size between 140 and 170 bp) is "trimmed" down to a discrete core of 140 bp. The "trimming" of monomers to 140 bp results from a 3' exonucleolytic digestion accompanied by a 5' clipping activity which is specific for the conformation of internucleosomal DNA. At higher concentrations of Exo III, the enzyme digests the 140 bp "trimmed" nucleosome core from both 3' ends without associated 5' nuclease activity. Most striking is the observation that the fragments produced during such a digestion display discrete single-stranded lengths that are integer multiples of 10 bases. For some dimer nucleosomes, Exo III can digest as many as 200 bases from at least one 3' end and produce a 10 base interval ladder from about 400 bases down to 180 bases. This suggests that the enzyme can traverse the length of an entire nucleosome without destroying whatever structural features are necessary to produce a 10 base DNA ladder.

Chromatin↗
PubMed1988

New spectrophotometric method for continuous recording of the spleen exonuclease activity.

Some of the synthetic chromophoric substrates of various enzymes cannot be used for direct spectrophotometric recording of the reactions, when a difference between the pH optimum of the enzyme reaction and the pH of maximum absorption of the released chromophore exists. In the present paper we describe a new method for following the time course of the spleen exonuclease-catalyzed reaction with thymidine 3'-monophospho-p-nitrophenyl ester as a substrate, based on the difference obtained in the absorbency of the substrate and its products in the far UV (at 330 nm). This difference, not published before, permits direct spectrophotometric recording of the amount of the hydrolyzed chromophoric substrate in acidic pH, whereas the maximum absorption of the product as accepted in the literature, is in alkaline pH. The molar absorption coefficient of the measurement at pH 5.7 is determined to be epsilon = 522 M-1.mm-1.

Chromogenic Compounds↗
PubMed1987

Mutagenic DNA repair in Escherichia coli. XIII. Proofreading exonuclease of DNA polymerase III holoenzyme is not operational during UV mutagenesis.

We have introduced a mutD5 mutation (which results in defective 3'-5'-exonuclease activity of the epsilon proofreading subunit of DNA polymerase III holoenzyme) into excision-defective Escherichia coli strains with varying SOS responses to UV light. MutD5 increased the spontaneous mutation frequency in all strains tested, including recA430, umuC122::Tn5, and umuC36 derivatives. It had no effect on UV mutability or immutability in any strain or on misincorporation revealed by delayed photoreversal in UV-irradiated umuC36, umuC122::Tn5, or recA430 bacteria. It is concluded that the epsilon proofreading subunit of DNA polymerase III holoenzyme is excluded, inhibited, or inoperative during misincorporation and mutagenesis after UV.

Arginine↗
PubMed1980

Cloning of the exonuclease III gene of Escherichia coli.

Overproducers of exonuclease III (exo III) were found within a colony bank containing ColE1-Escherichia coli hybrid plasmids. Through the enzymatic ligation of restriction enzyme fragments, the exo III gene, xth, was transferred to a thermoinducible, integration-proficient lambda phage and to a chimeric ColE1-lambda plasmid that was thermoinducible for lambda-directed DNA replication. Transfer of the xth gene was facilitated by a technique involving prior selection for Tn5 insertions into plasmid, thereby linking the gene to additional restriction sites and to a selectable (drug resistance) marker. After heat induction, cells bearing the thermoinducible ColE1-lambda-xth plasmid produced 120-fold more exo III than did plasmid-free cells. Enzyme production was not further enhanced by any of the following chromosomal mutations: dnaA, recBC, tob, or nusA snu. Several observations suggested that enzyme over-synthesis was the result primarily of lambda-detected replication rather than lambda-directed transcription.

Bacteriocin Plasmids↗
PubMed2004

Resistance towards exonucleases of dinucleotides with stereochemically altered internucleotide phosphate bonds.

Kinetic constants for the hydrolytic susceptibility of the internucleotide phosphate bond in normal dinucleotides [e.g., 2'-deoxycytidylyl-(3'>5')-2'-deoxyuridine (dCpdU) and 2'-deoxyadenylyl-(3'-->5')-2'-deoxycytidine (dApdC)] and isomeric dinucleotides [e.g., 2'-deoxycytidylyl-(3'-->5')-1'-deoxy-2'-isouridine (dCpisodU) and 1'-deoxy-2'-isoadenylyl-(3'-->5')-2'-deoxycytidine (isodApdC)], toward 5'- and 3'-exonucleases, phosphodiesterase I (PDE I) and phosphodiesterase II (PDE II) were experimentally determined and remarkable differences emerged. The study is of importance in the discovery of nuclease-stable inhibitors of HIV integrase, but may also have ramifications in the area of anti-sense oligonucleotides of therapeutic interest.

Dinucleoside Phosphates↗