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PubMed1997

Reversible inhibition of lambda exonuclease with high pressure.

The hydrolytic activity of lambda exonuclease, a highly processive enzyme, is inhibited by high pressure. When assayed at 67 MPa, the activity is 87% of the atmospheric rate; increasing the pressure to 336 MPa causes a greater than 99% inhibition of the enzyme activity. Decreasing the pressure from 336 MPa to 67 MPa at 20 degrees C reverses the inhibition. The use of hydrostatic pressure to control the activity of lambda exonuclease has potential practical application in DNA sample preparation, analysis, and sequencing.

Atmospheric Pressure↗
PubMed1998

Structural principles for the inhibition of the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioates.

A two-metal-ion catalytic mechanism has previously been proposed for several phosphoryl-transfer enzymes. In order to extend the structural basis of this mechanism, crystal structures of three single-stranded DNA substrates bound to the 3'-5' exonucleolytic active site of the large fragment of DNA polymerase I from Escherichia coli have been elucidated. The first is a 2.1 A resolution structure of a Michaelis complex between the large fragment (or Klenow fragment, KF) and a single-stranded DNA substrate, stabilized by low pH and flash-freezing. The positions and identities of the catalytic metal ions, a Zn2+ at site A and a Mg2+ at site B, have been clearly established. The structural and kinetic consequences of sulfur substitutions in the scissile phosphate have been explored. A complex with the Rp isomer of phosphorothioate DNA, refined at 2.2 A resolution, shows Zn2+ bound to both metal sites and a mispositioning of the substrate and attacking nucleophile. The complex with the Sp phosphorothioate at 2. 3 A resolution reveals that metal ions do not bind in the active site, having been displaced by a bulky sulfur atom. Steady-state kinetic experiments show that catalyzed hydrolysis of the Rp isomer was reduced only about 15-fold, while no enzyme activity could be detected with the Sp phosphorothioate, consistent with the structural observations. Furthermore, Mn2+ could not rescue the activity of the exonuclease on the Sp phosphorothioate. Taken together, these studies confirm and extend the proposed two-metal-ion exonuclease mechanism and provide a structural context to explain the effects of sulfur substitutions on this and other phosphoryl-transfer enzymes. These experiments also suggest that the possibility of metal-ion exclusion be taken into account when interpreting the results of Mn2+ rescue experiments.

Catalysis↗
PubMed2002

Determinants in nuclease specificity of Ape1 and Ape2, human homologues of Escherichia coli exonuclease III.

Abasic sites and non-conventional 3'-ends, e.g. 3'-oxidized fragments (including 3'-phosphate groups) and 3'-mismatched nucleotides, arise at significant frequency in the genome due to spontaneous decay, oxidation or replication errors. To avert the potentially mutagenic or cytotoxic effects of these chromosome modifications/intermediates, organisms are equipped with apurinic/apyrimidinic (AP) endonucleases and 3'-nucleases that initiate repair. Ape1, which shares homology with Escherichia coli exonuclease III (ExoIII), is the major abasic endonuclease in mammals and an important, yet selective, contributor to 3'-end processing. Mammals also possess a second protein (Ape2) with sequence homology to ExoIII, but this protein exhibits comparatively weak AP site-specific and 3'-nuclease activities. Prompted by homology modeling studies, we found that substitutions in the hydrophobic pocket of Ape1 (comprised of F266, W280 and L282) reduce abasic incision potency about fourfold to 450,000-fold, while introduction of an ExoIII-like pocket into Ape2 enhances its AP endonuclease function. We demonstrate that mutations at F266 and W280 of Ape1 increase 3' to 5' DNA exonuclease activity. These results, coupled with prior comparative sequence analysis, indicate that this active-site hydrophobic pocket influences the substrate specificity of a diverse set of sequence-related proteins possessing the conserved four-layered alpha/beta-fold. Lastly, we report that wild-type Ape1 excises 3'-mismatched nucleotides at a rate up to 374-fold higher than correctly base-paired nucleotides, depending greatly on the structure and sequence of the DNA substrate, suggesting a novel, selective role for the human protein in 3'-mismatch repair.

Amino Acid Sequence↗
PubMed1998

The exonuclease activity of HSV-1 UL12 is required for in vivo function.

The herpes simplex virus type 1 (HSV-1) UL12 gene encodes an alkaline pH-dependent deoxyribonuclease termed alkaline nuclease. A recombinant UL12 knockout mutant, AN-1, is severely compromised for growth, and analysis of this mutant suggests that UL12 plays a role in processing complex DNA replication intermediates (R. Martinez, R. T. Sarisky, P. C. Weber, and S. K. Weller, (1996) J. Virol. 70, 2075-2085). This processing step may be required for the generation of capsids that are competent for egress from the nucleus to the cytoplasm. In this report, we address the question of whether the AN-1 growth phenotype is due to the loss of UL12 catalytic activity. We constructed two point mutations in a highly conserved region (motif II) of UL12 and purified wild-type and mutant enzymes from a baculovirus expression system. Both mutant proteins are stable, soluble, and competent for correct nuclear localization, suggesting that they have retained an intact global conformation. Neither mutant protein, however, exhibits exonuclease activity. In order to examine the in vivo effects of these mutations, we determined whether expression of mutant proteins from amplicon plasmids could complement AN-1. While the wild-type plasmid complements the growth of the null mutant, neither UL12 mutant can do so. Loss of exonuclease activity therefore correlates with loss of in vivo function.

Amino Acid Sequence↗
PubMed1983

DNA degradation, UV sensitivity and SOS-mediated mutagenesis in strains of Escherichia coli deficient in single-strand DNA binding protein: effects of mutations and treatments that alter levels of Exonuclease V or recA protein.

Certain strains suppress the temperature-sensitivity caused by ssb-1, which encodes a mutant ssDNA binding protein (SSB). At 42 degrees C, such strains are extremely UV-sensitive, degrade their DNA extensively after UV irradiation, and are deficient in UV mutability and UV induction of recA protein synthesis. We transduced recC22, which eliminates Exonuclease V activity, and recAo281, which causes operator-constitutive synthesis of recA protein, into such an ssb-1 strain. Both double mutants degraded their DNA extensively at 42 degrees C after UV irradiation, and both were even more UV-sensitive than the ssb-1 single mutant. We conclude that one or more nucleases other than Exonuclease V degrades DNA in the ssb recC strain, and that recA protein, even if synthesized copiously, can function efficiently in recombinational DNA repair and in control of post-UV DNA degradation only if normal SSB is also present. Pretreatment with nalidixic acid at 30 degrees C restored normal UV mutability at 42 degrees C, but did not increase UV resistance, in an ssb-1 strain. Another ssb allele, ssb-113, which blocks SOS induction at 30 degrees C, increases spontaneous mutability more than tenfold. The ssb-113 allele was transduced into the SOS-constitutive recA730 strain SC30. This double mutant expressed the same elevated spontaneous and UV-induced mutability at 30 degrees C as the ssb+ recA730 strain, and was three times more UV-resistant than its ssb-113 recA+ parent. We conclude that ssb-1 at 42 degrees C and ssb-113 at 30 degrees C block UV-induced activation of recA protease, but that neither allele interferes with subsequent steps in SOS-mediated mutagenesis.

Bacterial Proteins↗
PubMed1983

Enzymatic toxicogenation of "activated" cyclophosphamide by 3'-5' exonucleases.

3'-5' Exonucleases from various sources were found to toxicogenate 4-hydroxycyclophosphamide ("activated" cyclophosphamide) by splitting the oxazaphosphorinane ring and releasing an alkylating moiety and acrolein. Neither cyclophosphamide (CP) nor the deactivated metabolites of CP, 4-keto-CP and carboxyphosphamide nor 4-(S-ethanol)-sulfido-CP were attacked by 3'-5' exonucleases. DNA polymerases with proofreading activity, such as DNA polymerase I from E. coli or DNA polymerase delta from rabbit bone marrow, exhibited a tenfold higher specific activity with "activated" CP than "plain" 3'-5' phosphodiesterases such as snake venom phosphodiesterase or 3',5'cyclic AMP phosphodiesterase from bovine heart tissue. High levels of toxicogenating activity were estimated in peripheric human lymphocytes and tissues of lymphatic origin, suggesting that enzymatic toxicogenation plays a key role with respect to the cytotoxic specificity of "activated" CP.

Animals↗
PubMed2005

Detection of interaction of binding affinity of aromatic hydrocarbon receptor to the specific DNA by exonuclease protection mediated PCR assay.

A novel exonuclease protection mediated PCR assay (EPM-PCR) to detect the interaction of protein and DNA at a dioxin-responsive enhancer (DRE) upstream of the CYP1A1 gene in rat hepatic cytosol was established. A double-stranded DNA fragment containing two binding sites was designed and incubated with the aryl hydrocarbon receptor (AhR) transformed by 2,3,7,8-tetrachlorodibenzo-p dioxin (TCDD) to generate TCDD: AhR: DNA complex which could protect receptor-binding DNA against exonuclease II (Exo III) digestion. With Exo III treatment, free DNAs were digested and receptor-bound DNAs remained that could be amplified by PCR. By agarose gel electrophoreses a clear band (285bp) was detected using TCDD-treated sample, while nothing with control samples. To detect transformed AhR-DRE complex, 2 fmol DNAs and 3 ug cytosol proteins were found to be sufficient in the experiment. Compared with gel retardation assay, this new method is more sensitive for monitoring the Ah receptor-enhancer interaction without radioactive pollution.

Animals↗
PubMed1996

Over-expression of the NUD1-coded endo-exonuclease in Saccharomyces cerevisiae enhances DNA recombination and repair.

The NUD1(=NUC2) gene of Saccharomyces cerevisiae has been subcloned and over-expressed in multi-copy plasmids. Enhanced expression of this nuclear endo-exonuclease gene was confirmed by Northern hybridization (>10-fold increase), and increased enzymatic activity (2.4-fold increase) was demonstrated by direct immunological assay using antibody raised against the purified Neurospora crassa endo-exonuclease. We found that increased expression of NUD1 was associated with an increase in cell survival after irradiation treatment with gamma rays, and an increase in radiation-induced mitotic recombination frequencies between duplicated gene sequences. The results presented here are consistent with previous biochemical data and confirm a role for the NUD1 gene product in recombination/repair processes.

DNA Repair↗
PubMed1996

Requirement of S. pombe exonuclease II, a homologue of S. cerevisiae Sep1, for normal mitotic growth and viability.

Exonuclease II (ExoII) from Schizosaccharomyces pombe is a 5'-->3' single-stranded DNA exonuclease. We have cloned its gene, exo2, whose nucleotide sequence revealed that ExoII is a homologue of the multifunctional Saccharomyces cerevisiae Sep1 protein (also called Kem1, Xrn1, Rar5, Dst2). S. pombe exo2 null mutants were cold-sensitive for growth, had increased cell size at the restrictive temperature, were hypersensitive to the mitotic inhibitor thiabendazol and to caffeine, and died rapidly in stationary phase. Many of these phenotypes are similar to those of sep1 (kem1 or xrn1) mutants of S. cerevisiae. In contrast, the exo2 mutation had only a moderate effect on progression through meiosis and no significant effect on meiotic recombination. We discuss possible functions of the multifunctional ExoII protein.

Amino Acid Sequence↗
PubMed1998

Genetic analysis of the yeast NUD1 endo-exonuclease: a role in the repair of DNA double-strand breaks.

The deoxyribonucleases (DNases) have been shown genetically to be important in the vital processes of DNA repair and recombination. The NUD1 gene, which codes for an endo-exonuclease of Saccharomyces cerevisiae, was analyzed for its role in the DNA double-strand break (DSB) repair processes. While the nud1 strain is only slightly sensitive to ionizing radiation, expression of the HO-endonuclease to introduce a DSB at the MAT locus in that strain results in cell death. Cell survival is inversely proportional to the duration of HO-endonuclease expression. Analysis of the surviving colonies from the nud1 strain indicated that many of the survivors are sterile and that the proportion of these sterile survivors increases with the time of HO-endonuclease expression. On the other hand, the surviving colonies from the isogenic NUD1 strain are mating-proficient. Interestingly, double mutants of nud1 rad52 are more resistant to ionizing irradiation than the rad52 strain and have a cell-survival fraction of 32% for rad52-1 nud1 and 9% for rad52::URA3 nud1 following prolonged HO-endonuclease expression, indicating that nud1 has a suppressor effect on the DSB-induced lethality in rad52. Polymerase chain reaction analysis showed that many of the nud1 survivors contained small alterations within theMAT locus, suggesting that the survivors arose through the process of non-homologous end-joining. These results suggest that the endo-exonuclease acts at a DSB to promote DNA repair via the homologous recombination pathway.

DNA↗
PubMed1999

YNT20, a bypass suppressor of yme1 yme2, encodes a putative 3'-5' exonuclease localized in mitochondria of Saccharomyces cerevisiae.

Mutation of YME genes in yeast results in a high rate of mitochondrial DNA escape to the nucleus. The synthetic respiratory growth defect of yme1 yme2 yeast strains is suppressed by recessive mutations in YNT20. Inactivation of YNT20 creates a cold-sensitive respiratory growth defect that is more pronounced in a yme1 background and which is suppressed by yme2. Inactivation of YNT20 causes a qualitative reduction in the rate of mitochondrial DNA escape in yme1, but not yme2, strains, suggesting that YNT20 plays a role in the yme1-mediated mitochondrial DNA escape pathway. YNT20p is a soluble mitochondrial protein that belongs to a subfamily of putative 3'-5' exonucleases. Furthermore, conserved sequence elements in Yme2p suggest that this protein may also function as an exonuclease.

ATP-Dependent Proteases↗
PubMed2001

Stationary-phase mutations in proofreading exonuclease-deficient strains of the yeast Saccharomyces cerevisiae.

In order to understand the role of yeast polymerases in spontaneous mutagenesis in non-growing cells we have studied the effects of mutations that impair the 3'--> 5' exonuclease function of polymerases delta (pol3-01) and epsilon (pol2-4) on the spontaneous reversion frequency of the frameshift mutation his7-2 in cells starved for histidine. We showed that for each exonuclease-deficient mutant the rate of reversion per viable cell per day observed in stationary-phase cells remained constant up to the 9th day of starvation (while the number of viable cells dropped), and was very similar to that observed in the same mutants during the growth phase. These data suggest that both DNA polymerases are involved in the control of mutability in non-growing cells.

DNA Polymerase II↗
PubMed1983

Enhanced polypeptide synthesis programmed by linear DNA fragments in cell-free extracts lacking exonuclease V.

Preparation of an in vitro coupled transcription-translation system from E. coli strains lacking exonuclease V has greatly improved the system for use with added linear DNA fragments. In fact, in extracts of these mutants linear fragments are stable for several hours. However the cell extracts show a high level of endogenous background. To avoid this complication extracts were prepared at 30 degrees C from a mutant carrying a temperature-sensitive exonuclease V. Polypeptides coded by a specific DNA region, e.g., delineated by restriction endonuclease sites, can now be easily identified.

DNA, Bacterial↗
PubMed1984

The excision of AP sites by the 3'-5' exonuclease activity of the Klenow fragment of Escherichia coli DNA polymerase I.

The 3' AP endonucleases (class I) are said to hydrolyze the phosphodiester bond 3' to AP sites yielding 3'-OH and 5'-phosphate ends; on the other hand, the resulting 3' terminal AP site is not removed by the 3'-5' exonuclease activity of the Klenow fragment [1]. We show that AP sites in DNA are easily removed by the 3'-5' exonuclease activity of the Klenow fragment and that they are excised as deoxyribose-5-phosphate. It is suggested that the 3' AP endonucleases are perhaps not the hydrolases they are supposed to be.

DNA↗
PubMed1986

Cloning and DNA sequence of the 5'-exonuclease gene of bacteriophage T5.

The nucleotide sequence of the BalI-PstI fragment of T5 DNA, 1347 bp in length, coding for 5'-exonuclease (D15 gene), has been determined. A coding region of the gene contains 873 bp and is preceded by a typical Shine-Dalgarno sequence. The D15 gene belongs to a cluster, consisting of at least 3 genes, in which a termination codon of a preceding gene overlaps an initiation codon of the following one. The sequence contains an open reading frame for 291 amino acid residues. The molecular mass of the 5'-exonuclease calculated from the predicted amino acid sequence is 33 400 Da.

Amino Acid Sequence↗
PubMed1990

Effect of divalent and monovalent cations on calf thymus PCNA-independent DNA polymerase delta and its 3'----5' exonuclease.

Recent data suggest that DNA polymerases alpha and delta might have a coordinate functional role at the replication fork. In this communication we show that Mg2+ is likely the natural metal activator for both enzymes. Mn2+, a known mutagenic agent, is a competitive inhibitor of Mg2+ for DNA polymerase delta and acompetitive for DNA polymerase alpha. The 3'----5' exonuclease activity associated with DNA polymerase delta is not affected upon addition of Mn2+. Be2+, another mutagenic agent, on the other hand, has an inhibitory effect on the 3'----5' exonuclease, but not on the DNA polymerase delta. The data presented might explain the mutagenic and carcinogenic potential of these two divalent cations.

Animals↗
PubMed1989

The recB gene product is essential for exonuclease V-dependent DNA degradation in vivo.

The recB21 mutation abolishes the exonuclease activity of the RecBCD enzyme (exonuclease V) of Escherichia coli. This might be due to the polar effect of recB21 on expression of the recD gene, the product of which is an essential component of the RecBCD enzyme. To achieve synthesis of the recD gene product, the recD+ plasmid was introduced into the recB21 mutant. Degradation of the endogenous DNA damaged by gamma-rays and degradation of the DNA of a phage T4 gene 2 mutant were nevertheless abnormally small in this strain. Thus, the functional recB gene product is required for the degradative function of the RecBCD enzyme.

DNA Repair↗
PubMed1992

Restriction analysis of the prototype strain of enteric adenovirus type 41 using exonuclease III.

Enteric adenoviruses 40 and 41 (Ad40 and Ad41) are a prominent cause of gastroenteritis in young children. Diagnosis of these enteric types by conventional means is complicated by their fastidious growth characteristics. Enteric adenovirus growth was enhanced by cocultivation. Typing of enteric isolates currently entails analysis of the extracted viral DNA with restriction enzymes. Restriction endonuclease fragments of the Ad41 strain Tak genome were ordered by (i) double digestion, (ii) release of restriction fragments from plasmids containing 84% of the Ad41 genome in EcoRI fragments A, B and C, (iii) hybridization of Southern blotted Ad41 fragments with EcoRI fragment containing plasmids and various segments of the Ad2 genome, (iv) sequential reduction of the genome beginning with terminal restriction fragments with exonuclease III and S1 nuclease. The termini of adenovirus genomes are difficult to clone and the use of exonuclease III is a useful alternative to conventional restriction mapping. DNA restriction patterns, fragment sizes and restriction maps of the Ad4 1 strain Tak with enzymes BamHI, BglII, ClaI, EcoRI, HindIII, PstI, SalI, SmaI and XhoI are presented. Prototype strain restriction maps should enable better understanding of adenovirus type 41 and its epidemiology.

Adenoviruses, Human↗