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At least 145 records · Page 8Linked to original sources
PubMed1975

Action of exonuclease V (the recBC enzyme) on ultraviolet-irradiated DNA.

Exonuclease V (the recBC enzyme) of Escherichia coli can release pyrimidine dimers from ultraviolet-irradiated linear duplex DNA though it acts more slowly on irradiated DNA than on non-irradiated DAN. However, close circular lambda-dv DNA or phi X174 replicative form I DNA is not attacked by exonuclease V even though the DNA has been irradiated and treated with T4 endonuclease V to produce single-stranded breaks at the 5'-side of pyrimidine dimers. When irradiated circular DNA, previously nicked by T4 endonuclease V, is briefly exposed to elevated temperature, the DAN becomes susceptible to the action of exonuclease V, and pyrimidine dimers are selectively released. The increased susceptibility to exonuclease V may be resulted from locarized denaturation, or "fraying" of the 5'-termini at the nicks. The preferential release of pyrimidine dimers was observed when irradiated DNA, treated with T4 endonuclease V, was incubated with crude extracts of Escherichia coli. The activity was found in various strains defective in exonuclease V and/or DNA polymerase I.

Coliphages↗
PubMed2004

Inactivation of the 3'-5' exonuclease of the replicative T4 DNA polymerase allows translesion DNA synthesis at an abasic site.

Here, we have investigated the consequences of the loss of proof-reading exonuclease function on the ability of the replicative T4 DNA polymerase (gp43) to elongate past a single abasic site located on model DNA substrates. Our results show that wild-type T4 DNA polymerase stopped at the base preceding the lesion on two linear substrates having different sequences, whereas the gp43 D219A exonuclease-deficient mutant was capable of efficient bypass when replicating the same substrates. The structure of the DNA template did not influence the behavior of the exonuclease-proficient or deficient T4 DNA polymerases. In fact, when replicating a damaged "minicircle" DNA substrate constructed by circularizing one of the linear DNA, elongation by wild-type enzyme was still completely blocked by the abasic site, while the D219A mutant was capable of bypass. During DNA replication, the T4 DNA polymerase associates with accessory factors whose combined action increases the polymerase-binding capacity and processivity, and could modulate the behavior of the enzyme towards an abasic site. We thus performed experiments measuring the ability of wild-type and exonuclease-deficient T4 DNA polymerases, in conjunction with these replicative accessory proteins, to perform translesion DNA replication on linear or circular damaged DNA substrates. We found no evidence of either stimulation or inhibition of the bypass activities of the wild-type and exonuclease-deficient forms of T4 DNA polymerase following addition of the accessory factors, indicating that the presence or absence of the proof-reading activity is the major determinant in dictating translesion synthesis of an abasic site by T4 DNA polymerase.

Bacteriophage T4↗
PubMed1996

p53 Protein exhibits 3'-to-5' exonuclease activity.

Highly purified p53 protein from different sources was able to degrade DNA with a 3'-to-5' polarity, yielding deoxynucleoside monophosphates as reaction products. This exonuclease activity was dependent on Mg2+ and inhibited by addition of 5 mM nucleoside monophosphates. This exonuclease activity is intrinsic to the wild-type p53 protein: it copurified with p53 during p53 preparation; only purified wild-type p53, but not identically purified mutant p53 proteins displayed exonuclease activity; the exonuclease activity could be reconstituted from SDS gel-purified and urea-renatured p53 protein and mapped to the core domain of the p53 molecule; and finally, purified p53 protein could be UV-cross-linked to GMP. A p53-intrinsic exonuclease activity should substantially extend our view on the role of p53 as a "guardian of the genome."

Antibodies, Monoclonal↗
PubMed1993

DNA polymerase epsilon: aphidicolin inhibition and the relationship between polymerase and exonuclease activity.

Calf thymus DNA polymerase epsilon readily uses short, synthetic oligonucleotides as substrates for both polymerase and exonuclease activity. These substrates were used to examine the mechanism of inhibition by aphidicolin. Aphidicolin competes with each of the four dNTPs for binding to a pol epsilon.DNA complex. Importantly, aphidicolin binds equally well regardless of the identity of the next template base to be replicated (Ki approximately 0.6 microM). Hydrolysis of synthetic templates of defined sequence by the 3'-->5' exonuclease was examined. pol epsilon preferred to hydrolyze single-stranded DNA 3-fold better than double-stranded DNA (Vmax/KM), while under Vmax conditions single-stranded DNA was hydrolyzed 100-fold faster than double-stranded DNA. Aphidicolin did not inhibit exonuclease activity on single-stranded DNA; however, activity on double-stranded DNA was partially inhibited. Formation of an E.[template.primer].aphidicolin ternary complex inhibits exonuclease activity. However, even under conditions where the polymerase site is completely blocked by a template-primer, the exonuclease retains significant activity.

Animals↗
PubMed1982

Specific stimulation of the T7 gene 6 exonuclease by the phage T7 coded deoxyribonucleic acid binding protein.

Bacteriophage T7 codes for a single-stranded DNA binding protein. This protein is the product of gene 2.5 and has been found previously to stimulate specifically the activity of the phage-coded DNA polymerase. We report here that the T7 DNA binding protein also stimulates the activity of the phage-coded exonuclease. The gene 6 exonuclease is a double-stranded DNA specific 5'-exonuclease that has been implicated in destruction of bacterial DNA, removal of RNA primers during DNA replication, genetic recombination, and DNA maturation. The enzyme is markedly inhibited by physiological concentrations of NaCl. This inhibition, which is due to a marked reduction in the Vmax of the enzyme, can be largely overcome by the phage-coded DNA binding protein. This stimulation is specific since the Escherichia coli DNA binding protein is without effect. The stimulation by the binding protein is apparently not due to its coating of the 3' single-stranded tails generated during the digestion. Kinetic studies show that the stimulation is due to a combined effect on both the Km and Vmax of the exonuclease. These studies are consistent with a loose binding of the binding protein to either the DNA or the exonuclease.

DNA Helicases↗
PubMed1990

Reduction of the potent DNA polymerase III holoenzyme 3'----5' exonuclease activity by template-primer analogues.

The DNA polymerase III holoenzyme of Escherichia coli contains a potent 3'----5' exonuclease that removes the terminal nucleotide from a synthetic deoxyoligonucleotide primer with a half-life of approximately 2 s. Degradation of primers could not be effectively prevented by permitting the holoenzyme to "idle" at the primer terminus in the presence of limited deoxynucleoside triphosphates. To further characterize this exonuclease and to develop stable primers to facilitate experimental manipulations, we synthesized a series of twelve 25-mer oligonucleotides that differed only in the two 3'-terminal residues. The penultimate position contained either a CMP or a dCMP residue, while at the terminal position either AMP, dAMP, 2',3'-dideoxyAMP, cordycepin (3'-dAMP), dAMP alpha S, or 2',3'-dideoxyAMP alpha S was incorporated. No single change at either the 3'-penultimate or 3'-terminal positions resulted in a decrease in the exonuclease rate greater than 10-fold; however, combined changes at these two sites resulted in a strong synergistic effect. Placing a ribonucleotide at the penultimate position coupled by a phosphorothioate linkage to a terminal 2',3'-dideoxynucleotide reduced the rate of exonucleolytic activity almost 30,000-fold (half-life approximately 16 h). If only the ribonucleotide and phosphorothioate substitutions were made, a primer capable of being efficiently elongated was generated that exhibited a 500-fold increase in stability (half-life = 40 min). The elemental effect observed by substituting a nonbridging oxygen in the terminal phosphodiester bond for sulfur increased from 1.5 to 200 as other substitutions were made that decreased the exonuclease rate. This was consistent with a change in the rate-limiting step of the exonuclease reaction from a conformational change to the chemical step where the covalent bond is cleaved. At least part of this effect appears to be due to perturbations within the enzyme's active site and not solely due to changes in electrophilicity.

DNA Polymerase III↗
PubMed2001

DNase I footprinting and enhanced exonuclease function of the bipartite Werner syndrome protein (WRN) bound to partially melted duplex DNA.

Werner syndrome is a premature aging and cancer-prone hereditary disorder caused by deficiency of the WRN protein that harbors 3' -->5' exonuclease and RecQ-type 3' --> 5' helicase activities. To assess the possibility that WRN acts on partially melted DNA intermediates, we constructed a substrate containing a 21-nucleotide noncomplementary region asymmetrically positioned within a duplex DNA fragment. Purified WRN shows an extremely efficient exonuclease activity directed at both blunt ends of this substrate, whereas no activity is observed on a fully duplex substrate. High affinity binding of full-length WRN protects an area surrounding the melted region of the substrate from DNase I digestion. ATP binding stimulates but is not required for WRN binding to this region. Thus, binding of WRN to the melted region underlies the efficient exonuclease activity directed at the nearby ends. In contrast, a WRN deletion mutant containing only the functional exonuclease domain does not detectably bind or degrade this substrate. These experiments indicate a bipartite structure and function for WRN, and we propose a model by which its DNA binding, helicase, and exonuclease activities function coordinately in DNA metabolism. These studies also suggest that partially unwound or noncomplementary regions of DNA could be physiological targets for WRN.

Base Sequence↗
PubMed1992

DNA polymerase, RNA polymerase and exonuclease activities on a DNA sequence modified by benzo[a]pyrene diolepoxide.

Adducts produced by modification of DNA with benzo[a]pyrene diolepoxide (BPDE) are known to inhibit both DNA and RNA synthesis. This phenomenon has been used as a method for determining the distribution of carcinogen binding within defined DNA sequences. A critical comparison of different enzyme activities on adducted DNA is needed, since different enzymes may process adducted DNA differently. Thus, we compared blocks in DNA polymerase activity with that of an RNA polymerase and with an exonuclease at single base resolution. BPDE adducts blocked the progression of cloned T7 DNA polymerase (Sequenase) in a dose-dependent manner. Although the majority of these blocks were at one base prior to adducted guanines, we also observed some blocks opposite specific guanines, suggesting that in some sequences the polymerase inserted a base opposite the modified guanine. Digestion with T4 DNA polymerase (3'----5') exonuclease activity was also blocked in BPDE-adducted DNA; however, fragments produced by blocks in T4 exonuclease migrated two or more bases longer than the corresponding guanine. Mapping of adduct distributions using both Sequenase and T4 exonuclease gave similar results, demonstrating that a long tract of guanines was preferentially modified, and within a polyguanine sequence, the 5' guanines were more heavily modified than the 3' guanines. Transcription of adducted DNA by SP6 RNA polymerase was also inhibited in a dose-dependent manner. However, adducted bases which posed strong blocks to the DNA polymerase were not always strong blocks to the RNA polymerase. Thus, in terms of adduct distribution, Sequenase and T4 exonuclease provided more consistent results than the RNA polymerase, since blockage of the RNA polymerase correlated poorly with guanines.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1986

Characterization of 3'----5' exonuclease associated with DNA polymerase of silkworm nuclear polyhedrosis virus.

3'----5' Exonuclease specific for single-stranded DNA copurified with DNA polymerase of nuclear polyhedrosis virus of silkworm Bombyx mori (BmNPV Pol). BmNPV Pol has no detectable 5'----3' exonuclease activity on single-stranded or duplex DNA. Analysis of the products of 3'----5' exonucleolytic reaction showed that deoxynucleoside monophosphates were released during the hydrolysis of single-stranded DNA. The exonuclease activity cosedimented with the polymerase activity during ultracentrifugation of BmNPV Pol in glycerol gradient. The polymerase and the exonuclease activities of BmNPV Pol were inactivated by heat with nearly identical kinetics. The mode of the hydrolysis of single-stranded DNA by BmNPV Pol-associated exonuclease was strictly distributive. The enzyme dissociated from single-stranded DNA after the release of a single dNMP and then reassociated with a next polynucleotide being degradated.

Animals↗
PubMed2001

A functional interaction of Ku with Werner exonuclease facilitates digestion of damaged DNA.

Werner syndrome (WS) is a premature aging disorder where the affected individuals appear much older than their chronological age. The single gene that is defective in WS encodes a protein (WRN) that has ATPase, helicase and 3'-->5' exonuclease activities. Our laboratory has recently uncovered a physical and functional interaction between WRN and the Ku heterodimer complex that functions in double-strand break repair and V(D)J recombination. Importantly, Ku specifically stimulates the exonuclease activity of WRN. We now report that Ku enables the Werner exonuclease to digest through regions of DNA containing 8-oxoadenine and 8-oxoguanine modifications, lesions that have previously been shown to block the exonuclease activity of WRN alone. These results indicate that Ku significantly alters the exonuclease function of WRN and suggest that the two proteins function concomitantly in a DNA damage processing pathway. In support of this notion we also observed co-localization of WRN and Ku, particularly after DNA damaging treatments.

Adenine↗
PubMed2004

Strong positional preference in the interaction of LNA oligonucleotides with DNA polymerase and proofreading exonuclease activities: implications for genotyping assays.

The effect of locked nucleic acid (LNA) modification position upon representative DNA polymerase and exonuclease activities has been examined for potential use in primer extension genotyping applications. For the 3'-->5' exonuclease activities of four proofreading DNA polymerases (Vent, Pfu, Klenow fragment and T7 DNA polymerase) as well as exonuclease III, an LNA at the terminal (L-1) position of a primer is found to provide partial protection against the exonucleases of the two family B polymerases only. In contrast, an LNA residue at the penultimate (L-2) position generates essentially complete nuclease resistance. The polymerase active sites of these enzymes also display a distinct preference. An L-1 LNA modification has modest effects upon poly merization, but an L-2 LNA group slows dTTP incorporation somewhat while virtually abolishing extension with ddTTP or acyTTP terminators, even with A488L Vent DNA polymerase engineered for terminator incorporation. These observations on active site preference have been utilized to demonstrate two novel assays: exonuclease-mediated single base extension (E-SBE) and proofreading allele-specific extension (PRASE). We show that a model PRASE genotyping reaction with L-2 LNA primers offers greater specificity than existing non-proofreading assays, whether or not the non-proofreading reaction employs LNA-modified primers.

Alleles↗
PubMed2004

Anticarsia gemmatalis multicapsid nucleopolyhedrovirus v-trex gene encodes a functional 3' to 5' exonuclease.

The viral three-prime repair exonuclease (v-trex) gene of the Anticarsia gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV) is the first baculovirus gene to be described with significant homology to a 3' exonuclease. v-trex is an early gene that is expressed by AgMNPV from 3 h post-infection. In the present study, the AgMNPV v-trex ORF was cloned into the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) under the control of a polyhedrin promoter. The resulting virus produced an abundant, soluble protein that migrated with an apparent size of 23.7 kDa. The 3' to 5' exonuclease activity associated with this v-trex-expressing recombinant AcMNPV was 2000-fold above that of wild-type AcMNPV. This exonuclease activity was inhibited by EDTA and was activated in the presence of Mg2+ and, to a lesser extent, Mn2+. From these results, the AgMNPV v-trex gene is concluded to encode an independently active 3' to 5' exonuclease.

Amino Acid Sequence↗
PubMed1993

Proof-reading 3'-->5' exonucleases isolated from rat liver nuclei.

Mammalian nuclear DNA polymerases alpha and beta are known to be devoid of the editing 3'-->5' exonucleolytic activity. Presumably this activity could be effected by the exonucleases non-associated covalently with DNA polymerases. Two 3'-->5' exonucleases of 40 kDa and 50 kDa (exo-40 and exo-5) have been isolated from rat liver nuclei and purified to near homogeneity. They are shown to excise mismatched nucleotides from poly[d(A-T)] template, respectively, 10-fold and 2-fold faster than the matched ones. Upon addition of either of these exonucleases to the DNA polymerase alpha from rat liver or calf thymus, the fidelity of in-vitro reproduction of the primed DNA from bacteriophage phi X174 amber 3 is increased 5-10-fold, levels of exonuclease and DNA-polymerase activities being similar. Extrapolation of in-vitro DNA-replication fidelity to the cellular levels of activities of the exonucleases and the alpha-polymerase suggests that exonucleolytic proof-reading augments the accuracy of DNA synthesis by 2-3 orders of magnitude.

Animals↗
PubMed1976

A second form of the single-strand specific endonuclease of Neurospora crassa which is associated with a double-strand exonuclease.

A second form of single-strand specific endonuclease, which is stable to heating up to 74 degrees C and does not bind strongly to phosphocellulose, has been partially purified from extracts of mycelia of wild-type Neurospora crassa. The endonuclease is associated with an equally heat-stable exonuclease which degrades linear but not circular double-stranded DNA and does not attack double-stranded RNA. The exonuclease probably also degrades single-stranded DNA. Both endonuclease and exonuclease activities are inhibited by 0.1-0.5 mM ATP. The exonuclease is preferentially inhibited by a variety of agents and preferentially inactivated by trypsin. A DNA-unwinding activity has also been detected in the nuclease preparation. Protease(s) present in the nuclease preparation destroy the DNA-unwinding and exonuclease activities on incubation at 37 degrees C, but do not affect the endonuclease activity. However, the heat-stability and chromatographic properties of the endonuclease are affected by this treatment. The altered properties of the endonuclease are very similar to those of the single-strand specific endonuclease which has been previously described. The combined nuclease activities of the unaltered preparational make up a putative recombination nuclease of N. crassa.

Adenosine Triphosphate↗
PubMed2003

On-off regulation of 3' exonuclease excision to DNA polymerization by Exo+ polymerase.

The role of 3' exonuclease excision in DNA polymerization was evaluated in primer extensions using 3' allele-specific primers that had exonuclease-digestible and exonuclease-resistant 3' termini. With exonuclease-digestible unmodified 3' mismatched primers, the exo+ polymerase yielded template-dependent products. Using exonuclease-resistant 3' mismatched primers, no primer-extended product resulted from exo+ polymerase. As a control, polymerase without proofreading activity yielded primer-dependent products from 3' mismatched primers. These data indicated that a successful removal of the mismatch is required for DNA polymerization from the 3' mismatched primers by exo+ polymerase. In addition to the well-known proofreading from this mismatch removal, the premature termination in DNA polymerization, due to the failure of the efficient removal of the mismatched nucleotides, worked as an off-switch in maintaining the high fidelity in DNA replication from exo+ polymerase.

Base Sequence↗
PubMed1978

[Inhibitors of exonuclease A5].

Over 30 compounds resembling to or being structural elements of the minimal substrate of exonuclease A5 were tested for their ability to inhibit the reaction catalyzed by this nuclease. The compounds containing less than two phosphate groups were shown to possess a low inhibitory activity, if any. CDP, double-stranded DNA and nucleoside-3',5'-diphosphates (pNp) proved to be effective exonuclease A5 inhibitors. Pyrophosphate stimulated the reaction in the case of low molecular weight substrates only. For the inhibitory activity of pNp to occur, the intactness of the nucleoside moiety as a whole was shown to be necessary, the activity level depending on the structure of both the base and the sugar components. A competitive mechanism of the inhibitory action was demonstrated for pTp, pdCp, pdGp, pdAp and pUp and the Ki values were determined. The affinity for the inhibitor decreased in the following order: pdCp greater than or equal to pTp greater than pdGp greater than pdAp. Ki for pdCp and pTp were found to be approximately 3.10(-6) M. The investigation of the inhibition mechanism as well the determination of Ki were accomplished with the help of homogenous low molecular weight substrates--ApApA and the phosphoamide MeOPheNH(pdA)2. These were chosen after kinetic parameters determination of the hydrolysis of 22 exonuclease A5 substrates, predominatly of the RpNpN type. On the basis of data obtained the specificity of exonuclease A5 is also discussed. Possible usefulness of immobilized competitive inhibitors of the pNp type not only for single nuclease isolation but for the separation of a mixture of different nucleases is considered. This possibility is based on the almost universal inhibitory effect of pNp on different nucleases and at the same time on their different affinity for the enzymes. In particular, this approach might be useful for the elimination of exonucleases and some other nucleolytic enzymes from the preparations of endonucleases-restrictases.

Cytidine Diphosphate↗
PubMed1990

Substrate specificity of the exonuclease associated with calf DNA polymerase.

The digestion mechanism and substrate specificity of the 3' to 5' exonuclease associated with calf thymus DNA polymerase epsilon have been examined. The use of single-molecule mismatched DNA substrates has allowed further characterization of the structural substrate requirements of the nonprocessive exonucleolytic activity of DNA polymerase epsilon. The digestion characteristics of these substrates demonstrated that a single-stranded segment 5' to the double-stranded complementary region is not a prerequisite for efficient exonucleolytic degradation of the mismatched single-stranded segment at the 3' end of the molecule. In contrast to the known inhibitory effect of DNA polymerase activity at moderate concentration of monovalent ions, the distribution of digestive products was virtually unaffected by the addition of 80 mM KCl to the reaction. Aphidicolin, an inhibitor of DNA polymerase activity, also inhibits exonucleolytic activity on substrates containing a terminal mismatch, while little effect is observed for the digestion of single-stranded DNA substrates. However, if a long terminal mismatched DNA substrate is used to mimic the structure of a single-stranded DNA molecule, the extent of digestion is significantly decreased by the addition of aphidicolin. Inhibition of the digestion of single-stranded DNA by aphidicolin is also observed if a double-stranded complementary region with a 3' single-stranded DNA segment is added to the reaction. These results indicate that aphidicolin inhibits exonuclease activity by sequestering the enzyme to a portion of mismatched DNA molecules away from the site where the exonuclease must act. Additionally, they demonstrate that, although the polymerase and exonuclease active sites are structurally linked, polymerase function is not a necessary requirement for exonuclease function.

Animals↗
PubMed1987

Selective oxidation of the exonuclease domain of bacteriophage T7 DNA polymerase.

Bacteriophage T7 DNA polymerase, the product of gene 5 of the phage, has both polymerase and single-and double-stranded DNA 3'-to 5'-exonuclease activities. The exonuclease activities can be inactivated selectively by an oxidation reaction that requires molecular oxygen, a reducing agent, and iron at a concentration less than or equimolar to that of the gene 5 protein. Both exonuclease activities can be diminished by several thousandfold, with only a small decline in the polymerase activity. Escherichia coli thioredoxin, an accessory protein that binds tightly to the gene 5 protein and increases the processivity of the polymerization reaction, has no effect on the rate of oxidation. We propose that iron binds specifically to the exonuclease domain and, in the presence of molecular oxygen and a reducing agent, generates reactive oxygen species that selectively modify amino acid residues essential for the exonuclease activities.

Bacterial Proteins↗