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PubMed1977

Novel properties of Escherichia coli exonuclease III.

The specificity of hydrolysis of polynucleotide termini by Escherichia coli exonuclease III was studied with the use of oligothymidylate annealed to polydeoxyadenylate. The size of the products after 3' leads to 5'-hydrolysis of 5'-labeled substrate is temperature-dependent. At 25 degrees the enzyme can hydrolyze a polynucleotide chain up to the last 5'-terminal dinucleotide. A gradation of higher 5'-terminal oligonucleotides of defined chain lengths is produced after limit digestion by the enzyme when the temperature is raised between 25 degrees to 60 degrees. When the oligothymidylate was labeled at the 3'-ends with ribonucleotides, it was observed that exonuclease III can cleave a single or two consecutive ribonucleotides regardless of whether the ribonucleotides are base-paired or mismatched.

Deoxyribonucleases↗
PubMed1976

5' leads to 3'-Exonucleases of bacteriophage T4.

Two enzyme activities which release nucleotides preferentially from the 5' termini of DNA were found in T4-infected Escherichia coli. Since no corresponding activities were found in uninfected cells, the activities appeared to be induced by T4. Both activities are capable of excising pyrimidine dimers from ultraviolet-irradiated DNA which has been treated with T4 endonuclease V. One of the activities , referred to as T4 exonuclease B, was purified 400-fold from an extract of T4v 1- infected cells. The enzyme initiates hydrolysis of DNA specifically at the 5' termini to yield products which are mainly oligonucleotides of varying length. The hydrolysis reaction proceeds in a limited manner. The enzyme shows optimal activity at pH 7.0 and absolutely requires Mg2+. The molecular weight of the enzyme , as estimated by gel filtration, is approximately 35,000. Another activity, referred to as T4 exonuclease C, was purified 240-fold from the extract. This activity also excises pyrimidine dimers from ultraviolet-irradiated, incised DNA and releases nucleotides at 5' termini. It has a pH optimum at 7.5 and requires Mg2+. The molecular weight of the enzyme is approximately 20,000.

Coliphages↗
PubMed1984

Exonucleases participating in the 3'end turnover of tRNA in Xenopus laevis.

Nuclei and enucleated Xenopus laevis oocytes were used as a source of exonucleases that hydrolyze the 3'terminus sequence of tRNA. The majority of the total activity was found in the cytoplasm. Several forms of enzyme activity are obtained upon fractionation in S-200 column. Both nuclear and cytoplasmic activities display a preferential cleavage of the terminal AMP residue. Similar results are obtained in vivo by microinjection of tRNA labelled in the CCA sequence. Aminoacylation of tRNA, on the other hand, protects the terminal nucleotide against exonuclease activity.

Animals↗
PubMedDate not supplied

[Molecular mechanisms of stabilization and preparation of metabolic breaks in DNA strands in vivo. I. Tandem action of exonuclease V and DNA polymerase II].

Investigation of the formation of metabolic imbalance breaks in the DNA of thy- cells of E. coli during thymine starvation is described. The results of experiments indicate that two enzymes--exonuclease V and 3' exonuclease activity of DNA polymerase II take part in the formation of metabolic imbalance gaps. The manifestation of activity of the enzymes has a tandem character.

DNA Polymerase II↗
PubMed1983

Purification and properties of single strand DNA-binding endo-exonuclease of Neurospora crassa.

Single strand DNA-binding endo-exonucleases purified from mitochondria, vacuoles, or a mixture of these organelles had the same high specific single strand DNase activity (910 mumol of nucleotides/min/mg), and each contained a polypeptide of Mr = 31,000-33,000 which was found to be active by sodium dodecyl sulfate-DNA-gel electrophoresis. The properties of the three preparations were identical in all respects tested. The enzyme showed distributive endonuclease activity with single strand DNA, but processive exonuclease activity with double strand DNA. In the former case, 5'-phosphoryl-terminated fragments were released at early times, while in the latter case, short 5'-oligonucleotides (n = 2-4) were released. Both activities were dependent on Mg2+ (or Mn2+), but to different extents. In 0.1 mM Mg2+, superhelical bacteriophage phi X174 (replicative form (RF II) DNA and, at converted to relaxed circular (RF II) DNA and, at higher enzyme concentrations, to unit length linear (RF III) DNA. In 10 mM Mg2+, these same conversions took place rapidly, and the RF III DNA which formed was degraded to pieces shorter than unit length. At very low enzyme concentrations, long single strand tails and gaps were detected in bacteriophage T7 linear double strand DNA molecules.

Chromatography, Affinity↗
PubMed1976

Endonuclease II of Escherichia coli is exonuclease III.

Exonuclease III, a phosphatase-exonuclease specific for bihelical DNA, wn the preparation was endonuclease II, an activity specific for DNA that has been partially depurinated by treatment with methyl methanesulfonate. The two activities, which could not be separated by electrophoresis, by sedimentation, or by gel filtration, were associated with a single monomeric protein of 28,000 daltons. To explain how a relatively small protein could have such diverse activities, it is proposed that one site on the enzyme can recognize interstrand spaces created either by depurination or by spontaneous terminal unwinding of a DNA duplex.

Deoxyribonucleases↗
PubMed1994

A conserved 5' to 3' exonuclease activity in the yeast and human nucleotide excision repair proteins RAD2 and XPG.

Saccharomyces cerevisiae RAD2 protein and its human homolog xeroderma pigmentosum group G (XPG) protein function in the incision step of nucleotide excision repair of DNA damaged by ultraviolet light. Both RAD2 and XPG proteins have been shown previously to possess an endonuclease activity. Using DNA substrates labeled at either the 5' end or 3' end, we now demonstrate that RAD2 protein also digests both single-stranded and double-stranded DNAs exonucleolytically with a 5' to 3' directionality. A 5' to 3' exonuclease activity is also present in the XPG protein, indicating evolutionary conservation of this activity. The possible role of RAD2 and XPG 5' to 3' exonuclease activity in nucleotide excision repair is discussed.

Base Sequence↗
PubMed1994

A multifunctional exonuclease from vegetative Schizosaccharomyces pombe cells exhibiting in vitro strand exchange activity.

A 140-kDa polypeptide (p140) has been purified over 2000-fold from vegetative Schizosaccharomyces pombe cells using an assay of homologous pairing and strand exchange between linear double-stranded DNA (dsDNA) and circular single-stranded DNA (ssDNA) in vitro. Electron microscopic analysis of the reaction products showed displacement of one strand of the linear duplex DNA by the circular ssDNA molecule. In addition, the protein contained 5' to 3' exonuclease activity on ssDNA and dsDNA (with a 50-fold preference on the single-stranded substrate) as well as on single-stranded RNA. Furthermore, p140 was capable of renaturing complementary ssDNA as shown by S1 nuclease assays. p140 behaved like a monomer in solution under reaction conditions. Direct comparison of the biochemical properties, sequence analysis, and cross-reactivity to a monoclonal antibody suggests that p140 is probably identical with ExoII, purified from S. pombe meiotic cells as a ssDNA exonuclease (Szankasi, P., and Smith, G. R. (1992) Biochemistry 31, 6769-6773). Given the diverse activities of p140, the protein might be involved in DNA and/or RNA metabolism in vivo.

Amino Acid Sequence↗
PubMed1994

The calf 5'- to 3'-exonuclease is also an endonuclease with both activities dependent on primers annealed upstream of the point of cleavage.

The catalytic activity of the calf thymus 5'- to 3'-exonuclease was measured on substrates consisting of two primers annealed adjacent to each other on a template. Exonucleolytic degradation of the downstream primer is very slow if the primers are separated by a gap of one nucleotide or if no upstream primer is present. When only a nick separates the primers, degradation is rapid. This suggests that the nuclease is designed to work with calf DNA polymerases such that synthesis from an upstream primer creates the favored nuclease substrate. Nuclease action then destroys the substrate, but it is regenerated by further polymerization. This process, termed nick translation, is necessary for both DNA replication and repair. If the downstream primer has an unannealed 5'-region, that region is removed by an endonuclease activity residing in the same enzyme. Efficient endonuclease action also requires an upstream primer that is annealed such that its 3'-end is directly adjacent to the annealed region of the downstream primer. This reaction is likely to be important for removal of DNA segments that are damaged such that exonuclease cleavage of the damaged site is not possible.

Animals↗
PubMed1993

Poly(U)-binding protein inhibits Drosophila pre-5 S RNA 3'-exonuclease digestion.

A approximately 50-kDa protein binds specifically to the 3' terminus of 135-nucleotide Drosophila pre-5 S RNA. Unlabeled poly(U) competes out protein binding and stimulates the activity of a 3'-exonuclease, which eventually degrades the substrate to 120 nucleotides, the size of mature 5 S RNA. In its RNA binding and UV cross-linking properties, the endogenous poly(U)-binding protein resembles human La, an autoantigen that binds the U > 3 3' ends of vertebrate RNA polymerase III primary transcripts. This protein appears to inhibit a 3' exonuclease and could protect 5 S RNA for faithful processing and transport.

Animals↗
PubMed1993

Effect of reaction pH on the fidelity and processivity of exonuclease-deficient Klenow polymerase.

We have examined as a function of pH the fidelity of DNA synthesis catalyzed by the 3'-->5' exonuclease-deficient form of the Klenow fragment of Escherichia coli DNA polymerase I. Increasing the pH of in vitro gap-filling reactions from pH 6.2 through 9.8 (37 degrees C increased the frequency of base substitution and minus-one-base frameshift mutations 50- and 40-fold, respectively, as measured by reversion of a nonsense or frameshift mutation within the lacZ alpha gene of bacteriophage M13mp2. To understand the mechanisms of high fidelity at low pH, we have examined the biochemical events associated with DNA synthesis at pH 6.2 that might be responsible for the observed accuracy in vitro. We show that while the steady-state frequency of T.dGTP misinsertion at the lacZ alpha opal codon is 20-fold lower at pH 6.2 than at pH 7.6, pH-dependent changes in the frequencies of G-dATP and A-dCTP base misinsertions at the lacZ alpha nonsense codon are insufficient to explain the fidelity changes observed in the gap-filling assay. However, the efficiency of steady-state extension synthesis from template-primers containing 3'-terminal T.G, G.A, and A.C (template-primer) mispairs was reduced up to 160-fold at pH 6.2 relative to pH 7.6. Analyses of the processivity of DNA polymerization versus pH demonstrated that at low pH the termination probability was decreased at specific template positions. Concomitantly, at sites where the termination probability was lower at pH 6.2, a decreased error rate was observed for base substitution mutations at three template positions and for minus-one-base frameshift mutations at two homopolymeric sequences relative to pH 7.6. We suggest that the observed increase in error discrimination by the exonuclease-deficient Klenow polymerase results from altered template binding properties of the enzyme at pH 6.2.

Base Sequence↗
PubMed1976

Human correxonuclease. Purification and properties of a DNA repair exonuclease from placenta.

An exonuclease, which hydrolyzes single-stranded DNA, has been purified from human placenta. It initiates hydrolysis at both the 3' and 5' termini of such DNA with equal facility, yielding 5'-phosphorylated oligonucleotides averaging 4 nucleotides in length. These oligonucleotides are released from both termini at equal rates with the same size distribution. Although not detectably active against intact native DNA, this enzyme can initiate hydrolysis at single-stranded breaks, creating a gap 30 to 40 nucleotides long. If a pyrimidine dimer is adjacent to this break, the enzyme, by virtue of its action at internal phosphodiester bonds, can excise such dimers. We refer to this exonuclease as human correxonuclease because of its excision capability.

Alkaline Phosphatase↗
PubMed1999

Single-molecule DNA digestion by lambda-exonuclease.

We used a bead displacement sensor to determine the enzymatic shortening of individual molecules of unstained lambda-DNA attached to optically trapped beads. The setup has been described previously (Dapprich and Nicklaus: Bioimaging 6:25-32, 1998) and works by observing the change in position of a trapped bead depending on its viscous drag force during motion. The drag force of a naked bead increases with each attached DNA molecule to a characteristic level that depends on the length and the number of DNAs per bead. A single undigested DNA molecule on a bead will remain stable for extended periods and exhibit a constant drag force in flow. If lambda-exonuclease is added, the drag force decreases from the level for one strand of DNA on a bead to that of a naked bead in about 45 min. This result indicates that the digestion of native lambda-DNA by lambda-exonuclease occurs at an average rate of approximately 15-20 Hz.

Bacteriophage lambda↗
PubMed1988

Use of restriction endonucleases and exonuclease III to expose halogenated pyrimidines for immunochemical staining.

We describe an enzymatic procedure for exposure of single-stranded DNA (ssDNA) containing the halogenated pyrimidines (HdUrd) bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd) in single cells to antibodies that bind to HrdUrd only in ssDNA. Production of ssDNA was accomplished by digesting the DNA using either restriction endonucleases alone or endonucleases followed by exonuclease III. The enzymatic production of ssDNA was maximal when 0.1 N HCl or 0.1 M citric acid plus Triton X-100 was added to extract nuclear proteins prior to enzymatic denaturation. The restriction endonucleases Bam HI, Dde I, Eco RI, and Hind III produced significant ssDNA when used alone to allow binding of detectable amounts of the anti-HdUrd antibody IU-4 in Chinese hamster ovary cells labeled with 10 microM BrdUrd or 10 microM IdUrd. However, these treatments did not expose sufficient ssDNA to allow binding of IU-1, an anti-HdUrd antibody with lower binding affinity. IU-4 binding was most intense after treatment with Eco RI. Treatment with exonuclease III following endonuclease digestion allowed substantially more IU-4 binding.

Animals↗
PubMed1991

Structural basis for the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism.

The refined crystal structures of the large proteolytic fragment (Klenow fragment) of Escherichia coli DNA polymerase I and its complexes with a deoxynucleoside monophosphate product and a single-stranded DNA substrate offer a detailed picture of an editing 3'-5' exonuclease active site. The structures of these complexes have been refined to R-factors of 0.18 and 0.19 at 2.6 and 3.1 A resolution respectively. The complex with a thymidine tetranucleotide complex shows numerous hydrophobic and hydrogen-bonding interactions between the protein and an extended tetranucleotide that account for the ability of this enzyme to denature four nucleotides at the 3' end of duplex DNA. The structures of these complexes provide details that support and extend a proposed two metal ion mechanism for the 3'-5' editing exonuclease reaction that may be general for a large family of phosphoryltransfer enzymes. A nucleophilic attack on the phosphorous atom of the terminal nucleotide is postulated to be carried out by a hydroxide ion that is activated by one divalent metal, while the expected pentacoordinate transition state and the leaving oxyanion are stabilized by a second divalent metal ion that is 3.9 A from the first. Virtually all aspects of the pretransition state substrate complex are directly seen in the structures, and only very small changes in the positions of phosphate atoms are required to form the transition state.

Amino Acid Sequence↗
PubMed1993

On the activities of Escherichia coli exonuclease III.

Exonuclease III (Exo III) of Escherichia coli is a DNA-modifying enzyme very frequently used in molecular biology. The experiments described here were carried out with the aims of reliably controlling exonuclease activity and of learning more about the enzyme's specificities. The dependence of Exo III activity on factors such as temperature (including heat inactivation), the concentrations of Exo III and NaCl, and the concentration and shape of 3' termini was investigated. Double stranded DNA was found to be a competitive inhibitor of the enzyme activity. Some four nucleotide 3' protrusions were shown to be sensitive to Exo III digestion. The synchronism of deletion was also examined. Implications for the proposed mechanism of activity are discussed.

Base Sequence↗
PubMed2001

Lambda exonuclease-based subtractive hybridization approach to isolate differentially expressed genes from leaf cultures of Paulownia kawakamii.

Genes that are preferentially expressed in a particular developmental pathway can be isolated by subtractive hybridization (SH). We developed a PCR-based approach coupled with lambda exonuclease digestion that allows for generating single-stranded tester and driver nucleic acids suitable for SH starting from cDNA libraries. An efficient subtraction strategy was developed to overcome some of the problems in the previously described SH protocols, such as the need for large amounts of experimental tissue, RNase contamination during solution hybridization, and postsubtraction recovery of nucleic acids. We used this method to obtain cDNA corresponding to genes expressed during adventitious shoot regeneration from excised leaf cultures of the fast-growing tree Paulownia kawakamii. Over 36 cDNA clones were isolated and 1 of the differentially expressed clones codes for a leucine zipper transcription factor. This clone showed about sixfold higher level of expression in the shoot-forming tissues (tester) compared to that in the callus-forming tissues (driver) of Paulownia, suggesting that differentially expressed genes can be efficiently isolated using this simple lambda exonuclease-based subtractive hybridization method.

Amino Acid Sequence↗
PubMed1993

DNA binding and cleavage of a novel antitumor antibiotic dynemicin A: detection of binding and cleavage sites by exonuclease III digestion and alkali-induced cutting reactions.

We found here that dynemicin A effectively breaks DNA strands under alkaline pH condition. Binding of dynemicin A to double-stranded DNA clearly interrupts the digestion reaction of exonuclease III. The DNA association sites of dynemicin A correspond considerably well to its cleavage sites. Dynemicin A seems to intercalate preferentially into the relaxed region of the DNA double helix. On the other hand, the alkali-product of dynemicin A was chromatographically identified with dynemicin N, suggesting a DNA cleavage mechanism similar to the reductant- and light-induced activation systems of dynemicin A. In order to detect drug binding to the relaxation structure in the DNA duplex, the present exonuclease III-digestion-stop-sequence method is useful.

Anthraquinones↗