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PubMed1993

Expression of Drosophila Rrp1 protein in Escherichia coli. Enzymatic and physical characterization of the intact protein and a carboxyl-terminally deleted exonuclease-deficient mutant.

Drosophila Rrp1 protein purified from embryos has four tightly associated enzymatic activities: DNA strand transfer, single-strand DNA renaturation, 3'-exonuclease, and apurinic endonuclease. Copurifying with these activities is a single polypeptide that has an apparent M(r) of 105,000 when estimated by SDS-polyacrylamide gel electrophoresis. To determine if this polypeptide is sufficient for these activities, it has been overexpressed in Escherichia coli. In crude extracts of E. coli cells, an ATP-independent Mg(2+)-dependent strand transfer activity is observed upon activation of the promoter that drives expression of Rrp1. Rrp1 protein purified from induced E. coli cells has electrophoretic, chromatographic, and enzymatic properties similar to those of Drosophila Rrp1 protein. The carboxyl-terminal region of Rrp1 (amino acids 428-679) is homologous to E. coli exonuclease III. Rrp1 deleted for this region cannot carry out DNA strand transfer, but can renature complementary single-strand DNA. The strand transfer activity of this truncated protein can be restored if DNA 3'-exonuclease is provided in trans by pretreating the double-strand DNA substrate with E. coli exonuclease III. This demonstrates a likely role of the exonuclease in the in vitro DNA strand transfer reaction carried out by Rrp1 protein. Such a role is also suggested by an analysis of the polarity of the strand transfer reaction.

Animals↗

PubMed1976

[Some peculiarities of transfer RNAs hydrolysis by exonuclease A5].

Unusual kinetics of the hydrolysis of the baker's yeast total tRNA and tRNA1Val by exonuclease A5 was found (two stages of the reaction; low initial velocity and still lower velocity of the second reaction stage; high Km values). These peculiarities were shown to be due to the three-dimensional structure of the substrate, which makes a large part of the tRNA molecule resistant to the exonuclease A5 attack. The exhaustive tRNA hydrolysis observed in the presence of very large doses of exonuclease A5 may be explained by a slight (less than 1%) endo-RNAase contamination in the exonuclease A5 preparation. The hydrolysis conditions become optimal, i.e. the exhaustive hydrolysis proceeds most rapidly, when the amount of endo-RNAase admixture reaches 20--30%. The artificial enzyme mixture of such a composition is a convenient reagent for RNA and expecially for tRNA hydrolysis under mild conditions to 5'-mononucleotides. The considerable resistance of tRNA to pure exonuclease A5 action will make it possible porbably to use tRNA instead of circular nucleic acids for endonuclease admixtures estimation in enzyme preparations.

Exonucleases↗

PubMed1993

Genetic and biochemical studies of bacteriophage T4 DNA polymerase 3'-->5'-exonuclease activity.

DNA polymerase exonucleolytic proofreading is important in attaining high fidelity DNA replication. One of the most well characterized proofreading activities is the 3'-->5'-exonuclease activity of bacteriophage T4 DNA polymerase. We have used genetic analyses and protein sequence comparisons to Escherichia coli DNA polymerase I to identify amino acids in the N-terminal region of T4 DNA polymerase that are required for exonucleolytic proofreading. Mutant DNA polymerases with amino acid substitutions D112A/E114A, D219A, or D324A reduced 3'-->5'-exonuclease activity 10(2)-10(4)-fold in various in vitro assays and decreased DNA replication fidelity in vivo. DNA replication activity was also reduced for the exonuclease-deficient DNA polymerases in vitro and in vivo. Reduction in DNA replication appeared to be due primarily to the interdependence of T4 DNA polymerase replication and proofreading activities; T4 DNA polymerase requires 3'-->5'-exonuclease activity to repair primer termini that are not suitable substrates for extension. Observations reported here provide further evidence in support of the proposal that DNA polymerases have distinct 3'-->5'-exonuclease and polymerase active sites.

Bacteriophage T4↗

PubMed1993

An error-correcting proofreading exonuclease-polymerase that copurifies with DNA-polymerase-alpha-primase.

A DNA polymerase with a 3'-to 5'-exonuclease that copurified with polymerase-primase from calf thymus was purified and extensively characterized. Its exonuclease degraded single-stranded DNA from 3' to 5' in a strictly distributive manner. On synthetic template-primer junctions, 3'-terminal mispairs were excised with a 10- to 20-fold preference over correctly paired nucleotides. In comparison to the 3'- to 5'-exonuclease the DNA polymerase activity was rather low. The ratio of nucleotides incorporated to nucleotides excised was in the order of 1 to 3 nucleotide insertions per excision, suggesting that net forward DNA synthesis is not the primary role of this DNA polymerase. DNA synthesis was performed with a low processivity in the presence and absence of PCNA. Both the polymerase and exonuclease activities were inhibited to a comparable extent by AMP. Thus, the exonuclease-polymerase might represent a novel DNA polymerase that we tentatively designate as DNA polymerase zeta. Possible benefits of DNA polymerase zeta in the process of error correction and the apparent dichotomy of an built-in proofreading activity for the processive DNA polymerases gamma, delta, and epsilon and an obviously external proofreading function for the less processive animal cell DNA polymerases alpha and beta are discussed.

Animals↗

PubMed1992

Site-directed mutagenesis at the Exo III motif of phi 29 DNA polymerase; overlapping structural domains for the 3'-5' exonuclease and strand-displacement activities.

In this report we present the alignment of one of the most conserved segments (Exo III) of the 3'-5' exonuclease domain in 39 DNA polymerase sequences, including prokaryotic and eukaryotic enzymes. Site-directed substitutions of the two most conserved residues, which form the Exo III motif Tyr-(X)3-Asp of phi 29 DNA polymerase, did not affect single-stranded DNA binding, DNA polymerization, processivity or protein-primed initiation. In contrast, substitution of the highly conserved Tyr residue by Phe or Cys decreased the 3'-5' exonuclease activity to 7.5 and 4.1%, respectively, of the wild-type activity. Change of the highly conserved Asp residue into Ala resulted in almost complete inactivation (0.1%) of the 3'-5' exonuclease. In accordance with the contribution of the 3'-5' exonuclease to the fidelity of DNA replication, the three mutations in the Exo III motif (Y165F, Y165C and D169A) produced enzymes with an increased frequency of misinsertion and extension of DNA polymerization errors. Surprisingly, the three mutations in the Exo III motif strongly decreased (80- to 220-fold) the ability to replicate phi 29 DNA, this behaviour being due to a defect in the strand displacement activity, an intrinsic property of phi 29 DNA polymerase required for this process. Taking these results into account, we propose that the strand displacement activity of phi 29 DNA polymerase resides in the N-terminal domain, probably overlapping with the 3'-5' exonuclease active site.

Amino Acid Sequence↗

PubMed1984

Activated cyclophosphamide: an enzyme-mechanism-based suicide inactivator of DNA polymerase/3'-5' exonuclease.

DNA polymerase I from E. coli can toxify activated cyclophosphamide (CP) by means of the 3'-5' exonuclease activity associated with the enzyme. Acrolein and an alkylating moiety are released in the process. Preincubation of DNA polymerase I with activated CP for 15-60 min leads to an increasing inhibition of DNA polymerase activity, which can be prevented when preincubation of DNA polymerase I with activated CP is carried out in the presence of 5' AMP, a competitive inhibitor of the 3'-5' exonuclease subsite of the enzyme. This demonstrates that toxification of activated CP by the 3'-5' exonuclease subsite of DNA polymerase is a prerequisite for the inhibition of DNA polymerase activity. The kinetics and the degree of DNA polymerase inhibition suggest that the alkylating moiety rather than acrolein released from activated CP during toxification is responsible for the inhibition of DNA polymerase. DNA polymerase with associated 3'-5' exonuclease activity has also been isolated from eukaryotic cells, and toxification of activated CP by such an enzyme (DNA polymerase delta from rabbit bone marrow) has been shown previously. Thus we suggest that toxification of activated CP by DNA polymerases/3'-5' exonucleases present mainly in proliferating cells might lead to the specific alkylation of macromolecules involved in the cell proliferation process, such as the DNA polymerase subsite of these enzymes and probably also the DNA bound to the enzymes. The relatively high cancerotoxic selectivity and cytotoxic specificity of activated CP could be based on this specific enzyme-mediated alkylation.

Acrolein↗

PubMed1996

'External' proofreading of DNA replication errors and mammalian autonomous 3'-->5'exonucleases.

Mammalian nuclear DNA polymerases alpha and beta are known to be devoid of the editing 3'-->5' exonucleolytic activity. The base substitutions misinserted by these polymerases could be eliminated with two kinds of an 'external' proofreading carried out (1) by the 3'-->5' exonuclease function intrinsic to DNA polymerases delta and epsilon or/and (2) by the autonomous 3'-->5' exonucleases non-associated covalently with DNA polymerases. DNA polymerases delta and epsilon can be separated from autonomous 3'-->5' exonucleases by means of sedimentation. Ultracentrifugation of the nuclear extracts and cytosols from normal and regenerating rat liver as well as from total embryos has shown the bulk of the cellular 3'-->5' exonucleolytic activity is due to autonomous nucleases. Moreover, the level of such a specific activity correlates with the replicative status of the organs from adult animals: spleen > regenerating liver > normal liver > cardiac muscle > brain, maximum difference being an order of magnitude. In addition, autonomous exonucleases were shown to be the constituents of the multienzyme forms of DNA polymerases alpha and beta. Hence, autonomous 3'-->5' exonucleases seem to be the principal participants in an 'external' proofreading.

Animals↗

PubMed1990

Mechanism of exonuclease action of BAL 31 nuclease.

Two kinetically and molecularly distinct forms ('fast' (F) and 'slow' (S] of nuclease BAL 31 from Alteromonas espejiana effect the length reduction of linear duplex DNAs through a 3'----5'-directed exonuclease activity in conjunction with an endonuclease activity against the 5'-terminated single-stranded tails generated by the exonuclease activity. No evidence for a 5'----3' mode of exonuclease action was seen. Single-stranded DNA is degraded predominantly by the 3'----5' exonuclease action. There is a pronounced decrease, to roughly constant values, of the average lengths of the tails in partially digested duplexes at a constant extent of digestion with increasing nuclease concentration. This decrease correlates with an increasing extent of ligatability, in the absence of repair, under conditions favoring the joining of fully base-paired ends. The exonuclease action, at least against duplex substrates, is quasi-processive and removes approx. 18 and 28 nucleotides per productive enzyme-substrate encounter for the S and F species, respectively. The dependence on Ca2+ and Mg2+ concentrations of the activities has been determined.

Bacteriophages↗

PubMed2003

Human AP endonuclease possesses a significant activity as major 3'-5' exonuclease in human leukemia cells.

Apurinic/apyrimidinic (AP) endonuclease (Ape1) is the major cellular enzyme responsible for repairing AP-sites in DNA. It can cleave the DNA phosphodiester backbone immediately 5(') to an AP-site. Ape1 also shows 3(')-phosphodiesterase activity, a 3(')-phosphatase activity, and an RNaseH activity. However, regarding its exonuclease activity, it remains controversial whether human Ape1 may possess a 3(')-5(') exonuclease activity. During the course of study to search for the major nuclease activity to double-stranded DNA in human leukemia cells, we purified a 37 kDa Mg(2+)-dependent exonuclease from cytosolic fraction of human leukemia U937 cells. Surprisingly, this exonuclease is Ape1. We demonstrated for the first time that Ape1 possesses a significant activity as major 3(')-5(') exonuclease in human leukemia cells. In addition, we also observed that translocation of cytoplasmic Ape1 into nucleus occurs during DNA damage.

Cell Nucleus↗

PubMed2006

WRN exonuclease activity is blocked by DNA termini harboring 3' obstructive groups.

Reactive oxygen species, generated either by cellular respiration or upon exposure to environmental agents such as ionizing radiation (IR), attack DNA to form a variety of oxidized base and sugar modifications. Accumulation of oxidative DNA damage has been associated with age-related disease as well as the aging process. Single-strand breaks harboring oxidative 3' obstructive termini, e.g. 3' phosphates and 3' phosphoglycolates, must be removed prior to DNA repair synthesis or ligation. In addition, 3' tyrosyl-linked protein damage, resulting from therapeutic agents such as camptothecin (CPT), must be processed to initiate repair. Several nucleases participate in DNA repair and the excision of 3' obstructive ends. As the protein defective in the segmental progeroid Werner syndrome (WRN) possesses 3'-5' exonuclease activity, and Werner syndrome cells are hypersensitive to IR and CPT, we examined for WRN exonuclease activity on 3' blocking lesions. Moreover, we compared side-by-side the activity of four prominent human 3'-5' exonucleases (WRN, APE1, TREX1, and p53) on substrates containing 3' phosphates, phosphoglycolates, and tyrosyl residues. Our studies reveal that while WRN degrades 3' hydroxyl containing substrates in a non-processive manner, it does not excise 3' phosphate, phosphoglycolate, or tyrosyl groups. In addition, we found that APE1 was most active at excising 3' blocking termini in comparison to the disease-related exonucleases TREX1, WRN, and p53 under identical physiological reaction conditions, and that TREX1 was the most powerful 3'-5' exonuclease on undamaged oligonucleotide substrates.

Cells, Cultured↗

PubMed2001

Long and accurate PCR with a mixture of KOD DNA polymerase and its exonuclease deficient mutant enzyme.

DNA polymerase from Thermococcus kodakaraensis KOD1 (previously Pyrococcus sp. KOD1) is one of the most efficient thermostable PCR enzymes exhibiting higher accuracy and elongation velocity than any other commercially available DNA polymerase [M. Takagi et al. (1997) Appl. Environ. Microbiol. 63, 4504-4510]. However, when long distance PCR (>5 kbp) was performed with KOD DNA polymerase, amplification efficiency (product yield) becomes lower because of its strong 3'-5' exonuclease activity for proof-reading. In order to improve a target length limitation in PCR, mutant DNA polymerases with decreased 3'-5' exonuclease activity were designed by substituting amino acid residues in conserved exonuclease motifs, Exo I (Asp141-Xaa-Glu), Exo II (Asn210-Xaa-Xaa-Xaa-Phe-Asp), and Exo III (Tyr311-Xaa-Xaa-Xaa-Asp). Exonuclease activity and amplification fidelity (error rate) of the DNA polymerases were altered by mutagenesis. However, long and accurate PCR by a single-type of mutant DNA polymerase was very difficult. The wild-type DNA polymerase (WT) and its exonuclease deficient mutant (N210D) were mixed in different ratio and their characteristics in PCR were examined. When the mixed enzyme (WT and N210D) was made at the ratio of 1:40, long PCR (15 kbp) at lower mutation frequency could be efficiently achieved.

Amino Acid Sequence↗

PubMed2000

Autonomous 3'-->5' exonucleases can proofread for DNA polymerase beta from rat liver.

Autonomous 3'-->5'exonucleases are not bound covalently to DNA polymerases but are often involved in replicative complexes. Such exonucleases from rat liver, calf thymus and Escherichia coli (molecular masses of 28+/-2 kDa) are shown to increase more than 10-fold the accuracy of DNA polymerase beta (the most inaccurate mammalian polymerase) from rat liver in the course of reduplication of the primed DNA of bacteriophage phiX174 amber 3 in vitro. The extent of correction increases together with the rise in 3'-->5' exonuclease concentration. Extrapolation of the in vitro DNA replication fidelity to the cellular levels of rat exonuclease and beta-polymerase suggests that exonucleolytic proofreading could augment the accuracy of DNA synthesis by two orders of magnitude. These results are not explained by exonucleolytic degradation of the primers ("no synthesis-no errors"), since similar data are obtained with the use of the primers 15 or 150 nucleotides long in the course of a fidelity assay of DNA polymerases, both alpha and beta, in the presence of various concentrations of 3'-->5' exonuclease.

Animals↗

PubMed1994

Pre-steady-state kinetic analysis of sequence-dependent nucleotide excision by the 3'-exonuclease activity of bacteriophage T4 DNA polymerase.

The effects of local DNA sequence on the proofreading efficiency of wild-type T4 DNA polymerase were examined by measuring the kinetics of removal of the fluorescent nucleotide analog 2-aminopurine deoxynucleoside monophosphate (dAPMP) from primer/templates of defined sequences. The effects of (1) interactions with the 5'-neighboring bases, (2) base pair stability, and (3) G.C content of the surrounding sequences on the pre-steady-state kinetics of dAPMP excision were measured. Rates of excision dAPMP from a primer 3'-terminus located opposite a template T (AP.T base pair) increased, over a 3-fold range, with the 5'-neighbor to AP in the order C < G < T < A. Rates of removal of dAPMP from AP.X base pairs located in the same surrounding sequence increased as AP.T < AP.A < AP.C < AP.G, which correlates with the decrease in the stabilities of these base pairs predicted by Tm measurements. A key finding was that AP was excised at a slower rate when mispaired opposite C located next to four G.C base pairs than when correctly paired opposite T next to four A.T base pairs, suggesting that exonuclease mismatch removal specificities may be enhanced to a much greater extent by instabilities of local primer termini than by specific recognition of incorrect base pairs. In polymerase-initiated reactions, biphasic reaction kinetics were observed for the excision of AP within most but not all sequence contexts. Rates of the rapid phases (30-40 s-1) were relatively insensitive to sequence context. Rapid-phase rates reflect the rate constants for exonucleolytic excision of dAPMP from melted primer termini for both correct and incorrect base pairs and were roughly comparable to rates of removal of dAPMP from single-stranded DNA (65-80 s-1). Rates of the slow phases (3-13 s-1) were dependent on sequence context; the slow phase may reflect the rate of switching from the polymerase to the exonuclease active site, or perhaps the conversion of a primer/template terminus from an annealed to a melted state in the exonuclease active site. These data, using wild-type T4 DNA polymerase and two exonuclease-deficient T4 polymerases, support a model in which exonuclease excision occurs on melted primer 3'-termini for both mismatched and correctly matched primer termini, and where specificity favoring removal of terminally mismatched base pairs is determined by the much larger fraction of melted-out primer 3'-termini for mispairs compared to that for correct pairs.

2-Aminopurine↗

PubMed1983

Periodicity of exonuclease III digestion of chromatin and the pitch of deoxyribonucleic acid on the nucleosome.

Exonuclease III has previously been shown to pause about every 10 nucleotides along the 3' strands while it invades the nucleosome core. Here, the exact periodicity of this digestion, i.e., the spacing of the pauses, was determined. Results showed that the exonuclease digests the first 20 nucleotides at the edge of the nucleosome core with a periodicity of approximately 11 nucleotides; in contrast, DNA closer to the center of the particle is digested with a smaller periodicity of about 10 nucleotides. These figures differ from the known periodicity of DNase I digestion, approximately 10 and 10.5 nucleotides at the edge and in the center of the nucleosome, respectively. Moreover, as shown by sedimentations in sucrose gradients, the structure of the nucleosome does not appear to be significantly altered by the gradual destruction of its DNA moiety by the exonuclease. Such stability of the nucleosome, along with other complementary observations, indicates that the transition in the digestion periodicity of the exonuclease may not be the consequence of a structural rearrangement of the particle upon trimming. This transition may rather be ascribed to the properties of the native nucleosome and to the intrinsic mechanism of action of the enzyme. Finally, evidence is presented which suggests that the exonuclease 10-nucleotide periodicity of digestion of the inner region of the nucleosome reflects a 10 base pair/turn pitch of the DNA in that region.

Animals↗

PubMed1996

A helical arch allowing single-stranded DNA to thread through T5 5'-exonuclease.

THE 5'-exonucleases are enzymes that are essential for DNA replication and repair. As well as their exonucleolytic action, removing nucleotides from the 5'-end of nucleic acid molecules such as Okazaki fragments, many 5'-3'-exonucleases have been shown to possess endonucleolytic activities. T5 5'-3'-exonuclease shares many similarities with the amino terminal of eubacterial DNA polymerases, although, unlike eubacteria, phages such as T5, T4 and T7 express polymerase and 5'-exonuclease proteins from separate genes. Here we report the 2.5-A crystal structure of the phage T5 5'-exonuclease, which reveals a helical arch for binding DNA. We propose a model consistent with a threading mechanism in which single-stranded DNA could slide through the arch, which is formed by two helices, one containing positively charged, and the other hydrophobic, residues. The active site is at the base of the arch, and contains two metal-binding sites.

Binding Sites↗

PubMed2000

Substrate specificity of the p53-associated 3'-5' exonuclease.

p53 exhibits 3'-5' exonuclease activity and the significance of this biochemical function is currently not defined. In order to gain information about the potential role(s) of this exonuclease activity, recombinant and wild-type human p53 was examined for excision of nucleotides from defined synthetic DNA substrates. p53 removes nucleotides threefold faster from single-strand DNA than from DNA duplexes, exhibits a 1.5-fold preference for 3'-terminals of DNA that contain a single nucleotide mispair (mismatch) as compared to correctly paired DNA and efficiently excises nucleotides from 3'-ends of blunt and cohesive (staggered) DNA double-strand breaks. The p53 exonuclease is predominantly non-processive on DNA which is 17 nucleotides long (or shorter) and processive on the longer 30-mers. The processivity of nucleotide excision is decreased in the presence of 50 mM potassium phosphate and eliminated when full-length p53 is replaced with the core domain, comprised of amino acids 82-292. Photoaffinity labeling indicates that (1) p53 monomers, rather than dimers, bind to single-strand forms of these oligomers; (2) complexes between p53 and 30-mers are more stable than those formed with 17-mers. The stability of these complexes determines processivity during nucleotide removal and modulates the 3'-5' exonuclease activity of p53. The relevance of substrate specificity of the p53 exonuclease to DNA repair is discussed.

Animals↗

PubMed1986

Exonuclease III and endonuclease IV remove 3' blocks from DNA synthesis primers in H2O2-damaged Escherichia coli.

Escherichia coli deficient in exonuclease III (xth gene mutants) are known to be hypersensitive to hydrogen peroxide. We now show that such mutants accumulate many more DNA single-strand breaks than do wild-type bacteria upon exposure to H2O2. DNA isolated from H2O2-treated xth- cells contains strand breaks that do not efficiently support synthesis by E. coli DNA polymerase I, indicating the presence of blocking groups at the DNA 3' termini. Purified E. coli exonuclease III activates this blocked DNA to allow substantial synthesis by polymerase I in vitro. Another E. coli enzyme, endonuclease IV, also activates primers for DNA polymerase. Exonuclease III accounts for greater than 95% of the total activity in E. coli crude extracts for removal of 3'-terminal phosphoglycolaldehyde esters from model DNA substrates. Purified exonuclease III and endonuclease IV can each efficiently remove 3'-terminal phosphoglycolaldehyde in vitro. An important physiological function for exonuclease III is thus the activation of blocked 3' ends for DNA repair synthesis. Endonuclease IV can also initiate the repair of ruptured 3'-deoxyribose in DNA.

DNA Damage↗

PubMed1987

A cryptic proofreading 3'----5' exonuclease associated with the polymerase subunit of the DNA polymerase-primase from Drosophila melanogaster.

The DNA polymerase-primase from Drosophila lacks 3'----5' exonuclease activity. However, a potent exonuclease can be detected after separating the 182-kDa polymerase subunit from the other three subunits of the enzyme (73, 60, and 50 kDa) by glycerol gradient sedimentation in the presence of 50% ethylene glycol. The exonuclease activity cosediments with the polymerase subunit, suggesting that the two activities reside in the same polypeptide. The 3'----5' exonuclease excises mismatched bases at the 3' termini of primed synthetic and natural DNA templates. Excision of a mispaired base at the 3' terminus occurs at a 10-fold greater rate than excision of the correctly paired base. When replication fidelity is measured by the bacteriophage phi X174 am3 reversion assay, the isolated polymerase subunit is at least 100-fold more accurate than either the intact polymerase-primase or a complex of the 182- and 73-kDa subunits. These results suggest that the 3'----5' exonuclease functions as a proofreading enzyme during Drosophila DNA replication in vitro and very likely in vivo.

Animals↗