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PubMed2005

The exonuclease and host shutoff functions of the SOX protein of Kaposi's sarcoma-associated herpesvirus are genetically separable.

The Kaposi's sarcoma-associated herpesvirus (KSHV) SOX protein, encoded by ORF37, promotes shutoff of host cell gene expression during lytic viral replication by dramatically impairing mRNA accumulation. SOX is the KSHV homolog of the alkaline exonuclease of other herpesviruses, which has been shown to function as a DNase involved in processing and packaging the viral genome. Although the exonuclease activity of these proteins is widely conserved across all herpesviruses, the host shutoff activity observed for KSHV SOX is not. We show here that SOX expression sharply reduces the half-life of target mRNAs. Extensive mutational analysis reveals that the DNase and host shutoff activities of SOX are genetically separable. Lesions affecting the DNase activity cluster in conserved regions of the protein, but residues critical for mRNA degradation are not conserved across the viral family. Additionally, we present evidence suggesting that the two different functions of SOX occur within distinct cellular compartments-DNase activity in the nucleus and host shutoff activity in the cytoplasm.

Cell Line↗
PubMed2004

3'-End polishing of the kinetoplastid spliced leader RNA is performed by SNIP, a 3'-->5' exonuclease with a Motley assortment of small RNA substrates.

In all trypanosomatids, trans splicing of the spliced leader (SL) RNA is a required step in the maturation of all nucleus-derived mRNAs. The SL RNA is transcribed with an oligo-U 3' extension that is removed prior to trans splicing. Here we report the identification and characterization of a nonexosomal, 3'-->5' exonuclease required for SL RNA 3'-end formation in Trypanosoma brucei. We named this enzyme SNIP (for snRNA incomplete 3' processing). The central 158-amino-acid domain of SNIP is related to the exonuclease III (ExoIII) domain of the 3'-->5' proofreading epsilon subunit of Escherichia coli DNA polymerase III holoenzyme. SNIP had a preference for oligo(U) 3' extensions in vitro. RNA interference-mediated knockdown of SNIP resulted in a growth defect and correlated with the accumulation of one- to two- nucleotide 3' extensions of SL RNA, U2 and U4 snRNAs, a five-nucleotide extension of 5S rRNA, and the destabilization of U3 snoRNA and U2 snRNA. SNIP-green fluorescent protein localized to the nucleoplasm, and substrate SL RNA derived from SNIP knockdown cells showed wild-type cap 4 modification, indicating that SNIP acts on SL RNA after cytosolic trafficking. Since the primary SL RNA transcript was not the accumulating species in SNIP knockdown cells, SL RNA 3'-end formation is a multistep process in which SNIP provides the ultimate 3'-end polishing. We speculate that SNIP is part of an organized nucleoplasmic machinery responsible for processing of SL RNA.

Animals↗
PubMed2005

The 3'->5' exonuclease of Apn1 provides an alternative pathway to repair 7,8-dihydro-8-oxodeoxyguanosine in Saccharomyces cerevisiae.

The 8-oxo-7,8-dihydrodeoxyguanosine (8oxoG), a major mutagenic DNA lesion, results either from direct oxidation of guanines or misincorporation of 8oxodGTP by DNA polymerases. At present, little is known about the mechanisms preventing the mutagenic action of 8oxodGTP in Saccharomyces cerevisiae. Herein, we report for the first time the identification of an alternative repair pathway for 8oxoG residues initiated by S. cerevisiae AP endonuclease Apn1, which is endowed with a robust progressive 3'-->5' exonuclease activity towards duplex DNA. We show that yeast cell extracts, as well as purified Apn1, excise misincorporated 8oxoG, providing a damage-cleansing function to DNA synthesis. Consistent with these results, deletion of both OGG1 encoding 8oxoG-DNA glycosylase and APN1 causes nearly 46-fold synergistic increase in the spontaneous mutation rate, and this enhanced mutagenesis is primarily due to G . C to T . A transversions. Expression of the bacterial 8oxodGTP triphosphotase MutT in the apn1Delta ogg1Delta mutant reduces the mutagenesis. Taken together, our results indicate that Apn1 is involved in an S. cerevisiae 8-oxoguanine-DNA glycosylase (Ogg1)-independent repair pathway for 8oxoG residues. Interestingly, the human major AP endonuclease, Ape1, also exhibits similar exonuclease activity towards 8oxoG residues, raising the possibility that this enzyme could participate in the prevention of mutations that would otherwise result from the incorporation of 8oxodGTP.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1990

Endo-exonuclease of Aspergillus nidulans.

Endo-exonuclease (EE) has been found in both active and inactive, but trypsin-activatable, forms in Aspergillus nidulans. Active EE was present mainly in nuclei, mitochondria, and vacuoles, while trypsin-activatable EE was mainly in the cytosol. The active form accounts for over 90% of the neutral deoxyribonuclease activity extracted from mycelia. A single strand (ss) DNA-binding EE associated with a 28 kilodalton (kDa) polypeptide was partially purified and characterized. It was found to closely resemble, in size and enzymological properties, the ss-DNA-binding EE previously purified from Neurospora crassa. Aspergillus nidulans EE was also found to be immunochemically related to the N. crassa EE and, like that enzyme, was probably derived from a polypeptide of 90 kDa or larger through proteolysis during extraction and purification. It had divalent metal ion-dependent (Mg2+, Mn2+, or Zn2+) activity on both DNA and RNA, which ultimately produced small 5'-P-terminated oligonucleotides. The nuclease activity was mixed endo- and exo-nucleolytic with ss-DNA as substrate, but largely exonucleolytic with double strand (ds) DNA. Superhelical phi X-174 DNA was nicked by EE to form relaxed circular and then linear ds-DNA, which was rapidly degraded to shorter fragments. Linearized pBR322 DNA was extensively nicked internally under conditions where there was relatively low exonuclease activity, but this nicking required that 5'-P-termini be present on the linear ds-DNA. The levels of active EE found in extracts of two recombination-deficient mutants of A. nidulans, uvsC and uvsE, dit not differ significantly from those in extracts of the wild type.

Aspergillus nidulans↗
PubMed2004

Exonuclease activity and P nucleotide addition in the generation of the expressed immunoglobulin repertoire.

BACKGROUND: Immunoglobulin rearrangement involves random and imprecise processes that act to both create and constrain diversity. Two such processes are the loss of nucleotides through the action of unknown exonuclease(s) and the addition of P nucleotides. The study of such processes has been compromised by difficulties in reliably aligning immunoglobulin genes and in the partitioning of nucleotides between segment ends, and between N and P nucleotides. RESULTS: A dataset of 294 human IgM sequences was created and partitioned with the aid of a probabilistic model. Non-random removal of nucleotides is seen between the three IGH gene types with the IGHV gene averaging removals of 1.2 nucleotides compared to 4.7 for the other gene ends (p < 0.001). Individual IGHV, IGHD and IGHJ gene subgroups also display statistical differences in the level of nucleotide loss. For example, within the IGHJ group, IGHJ3 has average removals of 1.3 nucleotides compared to 6.4 nucleotides for IGHJ6 genes (p < 0.002). Analysis of putative P nucleotides within the IgM and pooled datasets revealed only a single putative P nucleotide motif (GTT at the 3' D-REGION end) to occur at a frequency significantly higher then would be expected from random N nucleotide addition. CONCLUSIONS: The loss of nucleotides due to the action of exonucleases is not random, but is influenced by the nucleotide composition of the genes. P nucleotides do not make a significant contribution to diversity of immunoglobulin sequences. Although palindromic sequences are present in 10% of immunologlobulin rearrangements, most of the 'palindromic' nucleotides are likely to have been inserted into the junction during the process of N nucleotide addition. P nucleotides can only be stated with confidence to contribute to diversity of less than 1% of sequences. Any attempt to identify P nucleotides in immunoglobulins is therefore likely to introduce errors into the partitioning of such sequences.

Base Composition↗
PubMed2004

Evidence for interplay among yeast replicative DNA polymerases alpha, delta and epsilon from studies of exonuclease and polymerase active site mutations.

BACKGROUND: DNA polymerase epsilon (Pol epsilon) is essential for S-phase replication, DNA damage repair and checkpoint control in yeast. A pol2-Y831A mutation leading to a tyrosine to alanine change in the Pol epsilon active site does not cause growth defects and confers a mutator phenotype that is normally subtle but strong in a mismatch repair-deficient strain. Here we investigate the mechanism responsible for the mutator effect. RESULTS: Purified four-subunit Y831A Pol epsilon turns over more deoxynucleoside triphosphates to deoxynucleoside monophosphates than does wild-type Pol epsilon, suggesting altered coordination between the polymerase and exonuclease active sites. The pol2-Y831A mutation suppresses the mutator effect of the pol2-4 mutation in the exonuclease active site that abolishes proofreading by Pol epsilon, as measured in haploid strain with the pol2-Y831A,4 double mutation. Analysis of mutation rates in diploid strains reveals that the pol2-Y831A allele is recessive to pol2-4. In addition, the mutation rates of strains with the pol2-4 mutation in combination with active site mutator mutations in Pol delta and Pol alpha suggest that Pol epsilon may proofread certain errors made by Pol alpha and Pol delta during replication in vivo. CONCLUSIONS: Our data suggest that Y831A replacement in Pol epsilon reduces replication fidelity and its participation in chromosomal replication, but without eliminating an additional function that is essential for viability. This suggests that other polymerases can substitute for certain functions of polymerase epsilon.

Binding Sites↗
PubMed2005

DNA repair protein: endo-exonuclease as a new frontier in cancer therapy.

DNA repair mechanisms are essential for cellular survival in mammals. A rapid repair of DNA breaks ensures faster growth of normal cells as well as cancer cells, making DNA repair machinery, a potential therapeutic target. Although efficiency of these repair processes substantially decrease the efficacy of cancer chemotherapies that target DNA, compromised DNA repair contributes to mutagenesis and genomic instability leading to carcinogenesis. Thus, an ideal target in DNA repair mechanisms would be one that specifically kills the rapidly dividing cancer cells without further mutagenesis and does not affect normal cells. Endo-exonucleases play a pivotal role in nucleolytic processing of DNA ends in different DNA repair mechanisms especially in homologous recombination repair (HRR) which mainly repairs damaged DNA in S and G2 phases of the cell cycle in rapidly dividing cells. HRR machinery has also been implicated in cell signaling and regulatory functions in response to DNA damage that is essential for cell viability in mammalian cells where as the predominant nonhomologous end-joining pathway is constitutive. Although HRR is thought to be involved at other stages of the cell cycle, it is predominant in growing phases (S and G2) of the cell cycle. The faster growing cells are believed to carryout more HRR in replicative stages of the cell cycle where homologous DNA is available for HRR. Targeting endo-exonucleases specifically involved in HRR will make the normal cells less prone to mutagenesis, rendering the fast growing tumor cells more susceptible to DNA-damaging agents, used in cancer chemotherapy.

Antineoplastic Agents↗
PubMed2006

Exonuclease-mediated ELISA-like assay for detecting DNA-binding activity of transcription factors: measurement of activated NF-kappaB.

This paper describes an exonuclease-mediated enzyme-linked immunosorbent assay (ELISA)-like assay (EMEA) for detecting the DNA binding activity of nuclear factor kappaB (NF-kappaB). For EMEA, a special double-stranded DNA (dsDNA)-coupled plate was first prepared by immobilizing a DNA probe on an N-oxysuccinimide ester-coated plate. The immobilized DNA probe, which was internally labeled with digoxigenin (DIG)-dT contained a NF-kappaB binding consensus sequence for capturing activated NF- kappaB in analyzed samples. For measurement, the plate was first incubated with a protein sample and then treated with exonuclease III to eliminate the probes not bound by NF-kappaB. Finally, the probes protected by NF-kappaB were colorimetrically detected by an alkaline phosphatase (AP)-conjugated anti-DIG antibody. The major advantage of EMEA is that it detects NF-kappaB without the need for NF-kappaB antibodies. EMEA may provide a general approach for assays of DNA sequence-specific transcription factors for which specific antibodies are unavailable, expensive, or of insufficient quality.

Base Sequence↗
PubMed1999

Purification, characterization and cDNA cloning of an endo-exonuclease from the basidiomycete fungus Armillaria mellea.

We have purified an endo-exonuclease from the fruiting body of the basidiomycete fungus Armillaria mellea by using an ethanol fractionation step, followed by two rounds of column chromatography. The enzyme had an apparent molecular mass of 17500 Da and was shown to exist as a monomer by gel-filtration analysis. The nuclease was active on both double-stranded and single-stranded DNA but not on RNA. It was optimally active at pH8.5 and also exhibited a significant degree of thermostability. Three bivalent metal ions, Mg2+, Co2+ and Mn2+, acted as cofactors in the catalysis. It was also inhibited by high salt concentrations: activity was completely abolished at 150 mM NaCl. The nuclease possessed both endonuclease activity on supercoiled DNA and a 3'-5' (but not a 5'-3') exonuclease activity. It generated 5'-phosphomonoesters on its products that, after a prolonged incubation, were hydrolysed to a mixture of free mononucleotides and small oligonucleotides ranging in size from two to eight bases. Elucidation of its N-terminal amino acid sequence permitted the cDNA cloning of the A. mellea nuclease via a PCR-based approach. Peptide mapping of the purified enzyme generated patterns consistent with the amino acid sequence coded for by the cloned cDNA. A BLAST search of the SwissProt database revealed that A. mellea nuclease shared significant amino acid similarity with two nucleases from Bacillus subtilis, suggesting that the three might constitute a distinct class of nucleolytic enzymes.

Amino Acid Sequence↗
PubMedDate not supplied

[Antimutagenic role of autonomous 3'-->5'-exonucleases].

Our own and literary data about antimutagenic role of autonomous 3'-->5'-exonucleases (AE) are analyzed. AE are not bound covalently to DNA polymerases but often involved in replicative complexes. Intracellular overproduction of AE in bacteria is accompanied with the sharp suppression of mutagenesis, whereas the inactivation of AE in bacteria and higher fungi results in the increase of mutation rates by 2-3 orders of magnitude. The addition of AE in biologically meaningful concentrations to DNA polymerases elevates substantially the accuracy of their work in vitro. In these cases, the reverse mutation rates were measured in the DNA from phage (X174 amber 3, whereas the direct mutation rates--in the DNA from phage M13mp2, both being used as primer-templates for DNA synthesis and then transfected into spheroplasts of Escherichia coli. The accuracy of action of nuclease-free DNA polymerases alpha and beta are shown to raise in the presence of AE by 2-3 orders, the accuracy of moderately processive DNA polymerase I--by 2 orders, the accuracy of highly processive DNA polymerase delta--by 5-10 times, though the latter 2 polymerases display and their own 3'-->5'-exonucleolytic activity. AE, involved in the multienzyme DNA polymerase complexes, augment the accuracy of complexes action by 5-10 times. The model of "external" corrective role of AE in DNA biosynthesis is proposed. Study of 30 objects from all 3 kingdoms of live beings (from archae- and eubacteria to mammalia including human) has shown that AE account, as minimum, from 30 to 90% of the total cellular 3'-->5'-exonucleolytic activity. So AE increase essentially the intracellular ratio of values of 3'-->5'-exonuclease to DNA polymerase activities in the very various representatives from a phylogenetic tree that results always in the augmentation of the accuracy of DNA biosynthesis.

Animals↗
PubMed1979

Deoxyribonucleic acid polymerase of bacteriophage T7. Characterization of the exonuclease activities of the gene 5 protein and the reconstituted polymerase.

Homogeneous gene 5 protein of bacteriophage T7, a subunit of T7 DNA polymerase, catalyzes the stepwise hydrolysis of single-stranded DNA in a 3' leads to 5' direction to yield nucleoside 5'-monophosphates. The gene 5 protein itself does not hydrolyze duplex DNA. However, in the presence of Escherichia coli thioredoxin, the host-specified subunit of T7 DNA polymerase, duplex DNA is hydrolyzed in a 3' leads to 5' direction to yield nucleoside 5'-monophosphates. The apparent Km for thioredoxin in the reaction is 4.8 x 10(-8) M, a value similar to that for the apparent Km of thioredoxin in the complementation assay with gene 5 protein to restore T7 DNA polymerase activity. Both exonuclease activities require Mg2+ and a sulfhydryl reagent for optimal activity, and both activities are sensitive to salt concentration. Deoxyribonucleoside 5'-triphosphates inhibit hydrolysis by both exonuclease activities; hydrolysis of single-stranded DNA by the gene 5 protein is inhibited even in the absence of thioredoxin where there is less than 2% active T7 DNA polymerase. E. coli DNA binding protein (helix destabilizing protein) stimulates the hydrolysis of duplex DNA up to 9-fold under conditions where the hydrolysis of the single-stranded DNA is inhibited 4-fold.

DNA Helicases↗
PubMedDate not supplied

[Selective inhibition of 3'-5'-exonuclease activity of DNA-polymerase I from Escherichia coli by a fluoride ion].

The effect of NaF on the enzymatic activities of the large fragment of E. coli DNA polymerase I (Klenow enzyme-KE) with different DNA-substrates was studied. It was shown that fluoride ion at concentrations of 5-10 mM efficiently inhibits the 3'----5' exonuclease activity of KE but does not affect the polymerase activity of the enzyme. Selective inhibition of the 3'----5' exonuclease activity of KE is Mg-dependent and is observed with double- or single-stranded DNAs. In reaction with the 14-mer oligonucleotide annealed with single-stranded phage M13 DNA the enzyme was found not only to perform the exonucleolytic hydrolysis of the primers but to catalyse also a limited elongation of some primers, adding a few nucleotide residues in the absence of exogenous dNTP. The primer elongation is inhibited by inorganic pyrophosphatase and is stimulated by micromolar concentrations of exogenous pyrophosphate thus suggesting a possible role of PPi contamination in dNTP generation via pyrophosphorolysis. Traces of precursors in DNA preparations obtained by generally employed methods may serve as another source of nucleotides for the primer elongation.

Autoradiography↗
PubMed1989

Exonuclease activity that degrades histone mRNA is stable when DNA or protein synthesis is inhibited.

The induction and repression of histone synthesis during the cell cycle are regulated, in part, by modulating histone mRNA stability. When DNA synthesis stops, histone mRNA seems to be destabilized, perhaps via an autoregulatory circuit triggered by cytoplasmic histones. We have used an in vitro mRNA decay system to determine whether differential histone mRNA turnover is linked to changes in the basal activity of cytoplasmic mRNA-degrading enzymes. The basal level of the polysome-associated exonuclease enzyme or enzymes that degrade histone mRNA was similar in untreated cells and in cells exposed to DNA or protein synthesis inhibitors. Histone mRNA decay was accelerated in reactions supplemented with histones and soluble cytoplasmic factor(s) (S130), but S130s from control and inhibitor-treated cells were indistinguishable in these assays. The data indicate that basal exonuclease activity is stable or constitutive. The putative factor(s) required for autoregulating histone mRNA decay also do not change appreciably when DNA or protein synthesis is inhibited. The implications of these results with regard to the autoregulation of histone mRNA turnover are discussed.

Cycloheximide↗
PubMed1987

Purification and characterization of an endo-exonuclease from Saccharomyces cerevisiae that is influenced by the RAD52 gene.

An endo-exonuclease has been purified from logarithmically growing cells of the yeast Saccharomyces cerevisiae. Identification and purification of this nuclease was facilitated by its being precipitable with an antibody raised against a previously described Neurospora crassa endo-exonuclease (Resnick, M. A., Chow, T. Y.-K. Nitiss, J., and Game, J. C. (1984) Cold Spring Harbor Symp. Quant. Biol. 49, 639-649 and T. Y.-K. Chow and M. A. Resnick (1988) Mol. Gen. Genet., in press). The enzyme which was purified to near homogeneity was composed of a molecular weight 72,000 monomer. The single-strand nuclease activity is endonucleolytic and nonprocessive, whereas the double-strand DNase activity is exonucleolytic and weakly processive. Both nuclease activities have a pH optimum of 7.5, require Mg2+ or Mn2+ but not Zn2+ or Ca2+, are not inhibited by ATP, and exhibit the same kinetics of heat inactivation. Although this protein is not the product of the RAD52 gene, the greatly reduced amounts in rad52 mutants implicate the enzyme in repair and recombination processes in both mitotic and meiotic cells.

Chromatography, Gel↗
PubMed1987

[Purification and various properties of exonuclease from bacteriophage T4].

Exonuclease A was isolated from bacteriophage T4-infected cells of E. coli. The molecular mass of the enzyme is approximately 42,000 Da, pH optimum is 7-8.5, pI is 4.05. The enzyme activity depends on Mg2+, the optimal concentration of Mg2+ being 1-5 mM. The enzyme splits one- and two-helical DNA in the direction of 3'----5' and is a deoxyribonuclease splitting 5'-deoxynucleotides. The enzyme shows a practically equal affinity for one and two-helical DNA. The Km value for one- and two-helical DNA is 10 +/- 1 and 11 +/- 1 pmole of chain DNA, respectively. The Vmax value for one- and two-helical DNA is 61 +/- 5 and 45 +/- 5 pmole of nucleotides per min. Exonuclease A may be used for preparing substrates for DNA-polymerase T4 and Klenow fragment, i.e., during labeling of DNA at 3'-ends.

Chromatography, Ion Exchange↗
PubMed1987

Properties of the exonuclease activity that degrades H4 histone mRNA.

We have described a cell-free system for studying mRNA degradation (Ross, J., and Kobs, G. (1986) J. Mol. Biol. 188, 579-593). Using that system we found that human H4 histone mRNA was degraded in a 3' to 5' direction by an exonucleolytic activity. Here we investigate several properties of the crude system and of the exonuclease. A RNase inhibitor, such as that from placenta, was required to block nonspecific ribonucleases and thereby to permit different mRNAs to be degraded at different rates. The histone mRNA exonuclease required divalent cation (magnesium) but not exogenously added ATP or GTP. It functioned efficiently at monovalent cation concentrations ranging from 0.5 to 200 mM. It was bound to ribosomes isolated from cells lysed in low salt buffers. However, it was eluted in active form from the ribosomes by exposing them to 0.3 M KCl. The enzyme rapidly degraded deproteinized, 32P-labeled histone mRNA prepared enzymatically.

Cell-Free System↗
PubMed1987

Substrate specificity of the exonuclease activity that degrades H4 histone mRNA.

We have investigated the substrate specificity of an exonuclease that degrades human H4 histone mRNA, using synthetic RNA templates incubated in a cell-free mRNA decay system (Ross, J., and Kobs, G. (1986) J. Mol. Biol. 188, 579-593). Five RNAs that lacked poly(A), including histone, were degraded rapidly in vitro. Polyadenylated histone mRNA was degraded at least 10-fold more slowly than unmodified histone mRNA. Double-stranded RNA and DNA were very stable. Single-stranded DNA was degraded approximately 20-fold more slowly than single-stranded, non-polyadenylated RNA, and RNA with a 3' phosphoryl group was degraded more slowly than RNA with a 3'-hydroxyl group. Uncapped RNAs were degraded rapidly in the unfractionated system but were stable in reactions containing a ribosomal high salt wash extract. Therefore, the exonuclease activity released from ribosomes by high salt extraction was separated from the enzyme(s) that degraded uncapped RNAs.

DNA↗
PubMed1978

A DNase for apurinic/apyrimidinic sites associated with exonuclease III of Hemophilus influenzae.

An endonuclease purified from Hemophilus influenzae made single strand breaks in DNA containing apurinic or apyrimidinic sites but had no detectable endonuclease activity on untreated native DNA. The new 5'-termini created at the cleavage sites were base-free deoxyribose 5-phosphate residues. The enzyme preparation also catalyzed the exonucleolytic release of 5'-mononucleotides from bihelical DNA and the hydrolysis of DNA 3'-terminal phosphomonoesters. The phosphatase-exonuclease activity was indistinguishable from that reported by Gunther and Goodgal (J. Biol. Chem. (1970) 245, 5341-5349) and resembled that of exonuclease III of Escherichia coli. The endonucleolytic and exonucleolytic activities could not be separated by electrophoresis, sedimentation, or gel filtration, and they were also affected simultaneously by mutation. The enzymatic activities appear to be functions of a single monomeric protein (M(r) = 30,000).

Apurinic Acid↗