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PubMed1981

[Comparison of the molecular forms of exonuclease and ribonuclease in normal human spleen].

Molecular forms of exonuclease and ribonuclease from normal human spleen were separated by preparative electrofocusing and characterized by their enzymatic properties. Six molecular forms (E1 to E6) of exonuclease can be separated by isoelectric focusing from human normal spleen. Properties of these molecular forms have been studied: substrate specificity, hydrolysis of paranitrophenyl-thymidine-3'-monophosphate, optimum pH and heat-stability. So two enzymatic groups can be separated: - an acidic group contained forms E2, E3, E4 hydrolysing at high level the paranitrophenyl-thymidine-3'-monophosphate, with optimum pH between 5.0 and 5.5. These three forms were heat labile at 50 degrees C and belong probably to the same isoenzymatic group. - a neutral or more alkaline group was represented by the forms, E1, E5 and E6. These forms hydrolysed at very low level the paranitrophenyl-thymidine-3'-monophosphate and were different by their optimum pH and heat-stability. So, these three forms do not belong to the same isoenzymatic group. Profiles of ribonuclease showed three molecular forms R1, R2 and R3 different from exonucleases by their isoelectric point, optimum pH, substrate specificity and overall heat-stability. These ribonuclease forms differed from one another in optimum pH and probably do not belong to the same isoenzymatic group.

Chemical Phenomena↗
PubMed1982

Template-prime-dependent turnover of (Sp)-dATP alpha S by T4 DNA polymerase. The stereochemistry of the associated 3' goes to 5'-exonuclease.

T4 DNA polymerase converts (Sp)-2'-deoxyadenosine 5'-O-(1-thio[1-18O2]triphosphate) to 2'-deoxyadenosine 5'-O-[18O]-phosphorothioate in the presence of poly(d(A-T).poly(d(A-T)) template-primer. Control experiments involving either omitting the poly(d(A-T)).poly(d(A-T) template-primer or employing the (Rp)-2'-deoxyadenosine 5'-O-(1-thiotriphosphate) diastereomer showed no reaction. It is assumed, therefore, that this conversion as in the P--O case involves incorporation of the thionucleotide into the poly(d(A-T)) followed by hydrolysis resulting from the 3' goes to 5'-exonuclease activity. The 2'-deoxyadenosine 5'-O-[18O] phosphorothioate was converted to (Sp)-2'-deoxyadenosine 5'-O-(1-thio[1-18O]triphosphate), with no change in the configuration at P alpha by using the coupled adenylate kinase-pyruvate kinase enzyme system. A 31P NMR spectrum of the product showed that the 18O was entirely in the nonbridging position, indicating an overall retention in the net turnover process (i.e. incorporation followed by excision). Since the incorporation process involves an inversion of configuration around the phosphorus (Romaniuk, P. J., and Eckstein, F. (1982) J. Biol. Chem. 257, 7684-7688), it must be inferred that the 3' goes to 5'-exonuclease activity of T4 polymerase proceeds with inversion of configuration at the phosphorus atom, most simply via a direct displacement mechanism. This finding represents the first example of phosphodiester hydrolysis catalyzed by an exonuclease that does not involve a covalent phosphoryl-enzyme intermediate (Knowles, J. R. (1980) Annu. Rev. Biochem. 49, 877-919).

DNA-Directed DNA Polymerase↗
PubMed1982

Subunit structure of Escherichia coli exonuclease VII.

Exonuclease VII has been purified 7,500-fold to 87% homogeneity from Escherichia coli K12 using a new purification procedure. The enzyme has been shown to be composed of two nonidentical subunits of 10,500 and 54,000 daltons. This has been confirmed by restoration of exonuclease VII activity after renaturation of denatured and purified subunits. The structure of the native enzyme consists of one large subunit and four small subunits. We have previously isolated exonuclease VII mutant strains containing defects which map at two distinct loci. Subunit-mixing experiments utilizing wild type enzyme and temperature-sensitive enzyme produced by an xseB mutant strain have shown that the xseB gene codes for the small subunit of the enzymes.

Deoxyribonucleases↗
PubMed1986

A domain of the Klenow fragment of Escherichia coli DNA polymerase I has polymerase but no exonuclease activity.

The Klenow fragment of DNA polymerase I from Escherichia coli has two enzymatic activities: DNA polymerase and 3'-5' exonuclease. The crystal structure showed that the fragment is folded into two distinct domains. The smaller domain has a binding site for deoxynucleoside monophosphate and a divalent metal ion that is thought to identify the 3'-5' exonuclease active site. The larger C-terminal domain contains a deep cleft that is believed to bind duplex DNA. Several lines of evidence suggested that the large domain also contains the polymerase active site. To test this hypothesis, we have cloned the DNA coding for the large domain into an expression system and purified the protein product. We find that the C-terminal domain has polymerase activity (albeit at a lower specific activity than the native Klenow fragment) but no measurable 3'-5' exonuclease activity. These data are consistent with the hypothesis that each of the three enzymatic activities of DNA polymerase I from E. coli resides on a separate protein structural domain.

Binding Sites↗
PubMed1999

Characterization of a 3'-5' exonuclease associated with VDJP.

VDJP (V(D)J RSS Dependent DNA Joining Protein) was cloned based on binding to the nonamer portion of the V(D)J recombinational signal sequence (RSS), and genetic analysis revealed that VDJP is encoded by the same gene as the large subunit of Replication Factor C (RF-C). Recombinant VDJP has a site directed DNA joining activity and is capable of forming a covalent bond between DNA fragments containing an RSS element near their ends and exhibits 3' to 5' exonuclease activity. In this report, we examine the biochemical properties of the VDJP exonuclease activity such as directionality of nuclease action (3' to 5' or 5' to 3'), single-strand substrate preference, cleavage products, dependence on cofactors and metal cations, and optimal reaction conditions. From this analysis, we conclude that VDJP has an intrinsic 3'-5' exonuclease activity that produces mononucleotide products.

DNA↗
PubMed2000

Phage phi 29 DNA polymerase residues involved in the proper stabilisation of the primer-terminus at the 3'-5' exonuclease active site.

Three highly conserved amino acid residues have been characterised here as ssDNA ligands at the 3'-5' exonuclease active site of o29 DNA polymerase. The functional role of Tyr59, His61 and Phe69 residues of o29 DNA polymerase (belonging to Exo II motif, previously described as containing an invariant catalytic aspartate residue and two highly conserved ssDNA ligands) was assayed by biochemical analysis of six site-directed mutants at those residues. These studies revealed that the mutations introduced severely affected their ssDNA binding capacity and, as a consequence, the 3'-5' exonuclease activity on ssDNA substrates was also severely impaired, producing drastic defects in the maintenance of replication fidelity. Crystal structures of Klenow fragment of Pol Ik and Thermococcus gorgonarius DNA polymerase complexed with ssDNA at their 3'-5' exonuclease active sites revealed that residues Gln419 of the former, and Tyr209 of the latter, the counterparts of His61 of o29 DNA polymerase, are making contacts with the penultimate phosphodiester bond of ssDNA substrate. Here, the functional role of this residue is described.

Amino Acid Motifs↗
PubMed1999

Assays for analyzing exonucleases in vitro.

Ribonucleases play essential roles in cell growth, differentiation, and the response to stress. This article deals with exoribonucleases, enzymes that degrade RNAs beginning at either the 5' or 3' end and proceed down the length of the RNA. The preparation of a crude extract of a mammalian 3'-to-5' exonuclease is described. Assay conditions for both 5'-to-3' and 3'-to-5' exonucleases are given. One of these is a yeast enzyme that is known to be involved in mRNA decay. Others are vertebrate exonucleases that are presumed to have a role in mRNA stability but have not yet been proven to do so.

Animals↗
PubMed1984

Replacement synthesis labeling of DNA molecules in vitro using the Escherichia coli exonuclease III/DNA polymerase I enzyme pair.

In vitro labeling of DNA molecules using the Escherichia coli exonuclease III/DNA polymerase I enzyme pair has been examined as an alternative to existing methods of replacement synthesis labeling. It is shown that exonuclease III is able to act in a common restriction enzyme buffer [50 mM Tris (pH 8.0), 10 mM MgCl2, 50 mM NaCl] to produce a population of base-paired primer:template molecules which decrease uniformly in single-strand length with time. After heat inactivation of the exonuclease III and in the presence of radiolabeled deoxynucleotides the polymerase I reaction faithfully resynthesizes full-length molecules, asymmetrically labeled to high specific activity.

Base Composition↗
PubMed1985

Isolation and crystallization of lambda exonuclease.

lambda Exonuclease is a deoxyribonuclease induced by bacteriophage lambda. Mutations in the structural gene for the protein affect general recombination and indicate a possible function for the enzyme. A large scale isolation procedure was employed to purify enough enzyme from a heat-induced lambda lysogen for X-ray crystallographic analysis. Analytical ultracentrifugation and SDS-polyacrylamide electrophoresis revealed that lambda exonuclease is a tetramer with molecular mass 107,000 Da. Crystallization trials produced morphologically perfect crystals of a size suitable for X-ray diffraction studies. Cubic crystallographic symmetry was indicated by the lack of birefringence when the crystals were inspected with polarized light. X-ray precession photographs indicated that lambda exonuclease crystallizes in a space group of P4(1)32, or its enantiomorph P4(3)32, with 24 tetramers in the unit cell of edge 210 A.

Bacteriophage lambda↗
PubMed1990

NaF and mononucleotides as inhibitors of 3'-5'-exonuclease activity and stimulators of polymerase activity of E. coli DNA polymerase I Klenow fragment.

It has been shown that, in the absence of dATP in the poly(dT).oligo(dA) template-primer complex, the rate of primer cleavage by the E. coli DNA polymerase I Klenow fragment equals 4% of polymerization rate, while in the presence of dATP it equals as much as 50-60%. NaF and NMP taken separately inhibit exonuclease cleavage of oligo(dA) both with and without dATP. The addition of NaF (5-10 mM) or NMP (5-20 mM) increases the absolute increment of polymerization rate 5-9-fold relative to the absolute decrement of the rate of nuclease hydrolysis of primer. This proves the assumption that not more than 10-20% of primer molecules, interacting with the exonuclease center of polymerase, are cleaved by the enzyme. Presumably, NaF and nucleotides disturb the coupling of the 3'-end of oligonucleotide primer to the exonuclease center of the enzyme. As the primers mostly form complexes with the polymerizing center, the reaction of polymerization is activated.

DNA Polymerase I↗
PubMed1983

Characterization of human cytomegalovirus-induced DNA polymerase and the associated 3'-to-5', exonuclease.

A DNA polymerase activity induced by human cytomegalovirus (HCMV) was separated from host cell DNA polymerase and purified by phosphocellulose and DNA-cellulose column chromatography. The DNA polymerase activity was strongly inhibited by phosphonoacetic acid, aphidicolin, araATP, and N-ethylmaleimide, but it was resistant to 2',3'-dideoxyTTP. The sensitivity of HCMV-induced DNA polymerase to these reagents resembles that of host cell DNA polymerase alpha. However, HCMV-induced DNA polymerase activity was stimulated several fold by 100 mM ammonium sulfate, by which DNA polymerase alpha activity was strongly inhibited. Furthermore, it was found that a 3'-to-5' exonuclease activity was tightly associated with the HCMV-induced DNA polymerase. The exonuclease was also stimulated by ammonium sulfate, was inhibited by phosphoacetic acid, and it preferred single-stranded DNA as a substrate. The results suggest that the 3'-to-5' exonuclease may play a role in proofreading in the polymerization process as an integral part of the HCMV-induced DNA polymerase.

Ammonium Sulfate↗
PubMed1995

Inactivation of the 5'-3' exonuclease of Thermus aquaticus DNA polymerase.

The gene for Thermus aquaticus (Taq) DNA polymerase enzyme (Taq Pol I) was mutagenized and sixty-two candidate clones were screened for enzyme activity. Two of the clones expressed enzymes (*Taq-3 and *Taq-5) that showed very reduced 5'-3' exonuclease activity and normal DNA polymerase activity. These two enzymes showed heat resitance and storage stability similar to Taq Pol I and had similar effectiveness in PCR. Processivity of the polymerases was compared by measuring the extension of an end-labeled primer annealed to a single stranded DNA, as well as by a PCR method. The processivity of *Taq-3 and *Taq-5 was similar to Taq Pol I (50-80 nucleotides) and more processive than a Taq Pol I deficient in the 5'-3' exonuclease due to absence of the first 290 amino acids (Stoffel fragment). The results indicate two amino acids which are required for normal 5'-3' exonuclease activity in Taq Pol I (Arg-25 and Arg-74).

Cloning, Molecular↗
PubMed2006

The retinitis pigmentosa-mutated RP2 protein exhibits exonuclease activity and translocates to the nucleus in response to DNA damage.

Retinitis pigmentosa (RP) is a genetically heterogeneous disease characterized by degeneration of the retina. Mutations in the RP2 gene are linked to the second most frequent form of X-linked retinitis pigmentosa. RP2 is a plasma membrane-associated protein of unknown function. The N-terminal domain of RP2 shares amino acid sequence similarity to the tubulin-specific chaperone protein co-factor C. The C-terminus consists of a domain with similarity to nucleoside diphosphate kinases (NDKs). Human NDK1, in addition to its role in providing nucleoside triphosphates, has recently been described as a 3' to 5' exonuclease. Here, we show that RP2 is a DNA-binding protein that exhibits exonuclease activity, with a preference for single-stranded or nicked DNA substrates that occur as intermediates of base excision repair pathways. Furthermore, we show that RP2 undergoes re-localization into the nucleus upon treatment of cells with DNA damaging agents inducing oxidative stress, most notably solar simulated light and UVA radiation. The data suggest that RP2 may have previously unrecognized roles as a DNA damage response factor and 3' to 5' exonuclease.

Active Transport, Cell Nucleus↗
PubMed1997

Single-strand-specific DNase activity is an inherent property of the 140-kDa protein of the snake venom exonuclease.

Polyclonal antibodies against the exonuclease from Crotalus adamanteus venom (the 140-kDa protein) inhibit both the exonucleolytic and the single-strand-specific endonucleolytic activities, present in the exonuclease preparation. The antibodies also diminish the ability of the enzyme to split the negatively supercoiled Bluescript KS+ in the AT-rich fragment near-by the transcription termination site of the Ampicillin gene. Therefore the single-strand-specific endonucleolytic activity was attributed to the protein molecule of the exonuclease. The processivity of the exonucleolytic action was found to be less than 3 monomers as indicated by the heparin trapping method.

Animals↗
PubMed2003

The acetylatable lysines of human Fen1 are important for endo- and exonuclease activities.

Human Fen1 can be acetylated in vivo and in vitro resulting in reduced endonuclease and exonuclease activities in vitro. Acetylation occurs at four lysines located at the C terminus of Fen1, which is important for DNA binding. In this paper we show that Fen1 mutant proteins lacking the lysines at the C terminus have both reduced PCNA independent exonucleolytic and endonucleolytic activities. However, lysines at the C terminus are not required for PCNA stimulation of human Fen1. A double flap substrate was optimal for human Fen1 endonuclease and did not require the C-terminal lysines. Similarly, a one nucleotide 3'-overhang nick substrate was optimal for human Fen1 exonuclease and also did not require the C-terminal lysines. Finally, we found by an electromobility shift assay that human Fen1 had a different mode of binding with a double flap substrate containing a one nucleotide 3'-tail when compared to various other flap substrates. Taken together, our results confirm the double flap substrate as the likely in vivo intermediate for human Fen1 and that the C-terminal lysines are important for the endonuclease and exonuclease activities likely through DNA binding.

Acetylation↗
PubMed1998

Effect of a 3'-->5' exonuclease with a proofreading function on the fidelity of error-prone DNA polymerase alpha from regenerating liver of aged rats.

A nuclease that releases noncomplementary nucleotides from the 3'-end of DNA was isolated and highly purified from rat liver extract. The d(T9-C) priming activities for DNA synthesis in vitro by DNA polymerases alpha and beta were recovered by the addition of this enzyme, which itself does not contain a DNA polymerase activity. This nuclease hydrolysed nucleotides from the 3'-end, but did not remove [32P]-labeled nucleotides from the 5'-terminus of specifically labeled DNA. Also, the reaction products released from the 3'-end of DNA were all mononucleotides. These results indicate that the exonuclease is a 3'-->5' exonuclease with properties the same as those of DNase VII from human placenta. Rat DNase VII requires 4 mM MgCl2 or 0.125 mM MnCl2 for maximum activity, and shows a pH optimum of 7.5. These optimal conditions are similar to those of DNA polymerases, and indicate that both rat DNase VII and DNA polymerases are able to act under same conditions. Non-complementary nucleotide incorporation by DNA polymerase alpha from aged rat has been observed during in vitro DNA synthesis on poly dA-dT10. The amount of this mis-incorporation is decreased by the coexistence of the 3'-->5' exonuclease, but not all errors are edited out. Thus, this rat DNase VII is suggested to play an important role in proofreading during DNA synthesis.

Aging↗
PubMed1991

Role of divalent cations in the 3',5'-exonuclease reaction of DNA polymerase I.

X-ray studies of the proofreading 3',5'-exonuclease site of the large (Klenow) fragment of DNA polymerase I have detected a binuclear metal complex consisting of a pentacoordinate metal (site A) which shares a ligand, Asp-355, with an octahedral metal (site B) [Freemont, P. S., Friedman, J. M., Beese, L. S., Sanderson, M. R., & Steitz, T. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8924-8928; Beese, L. S., & Steitz, T. A. (1991) EMBO J. 10, 25-33]. Kinetic studies of the activation of the 3',5'-exonuclease reaction by Co2+, Mn2+, or Mg2+, at low concentrations of DNA, reveal sigmoidal activation curves for the three metal ions with Hill coefficients of 2.3-2.4 and K0.5 values of 16.6 microM, 4.2 microM, and 343 microM, respectively. The binding of Co2+ to the enzyme results in the appearance of an intense visible absorption spectrum of the metal ion with maxima at 633, 570, and 524 nm and extinction coefficients of 190, 194, and 150 M-1 cm-1, respectively, suggesting the formation of a pentacoordinate Co2+ complex. Optical titration with Co2+ yields a sigmoidal titration curve which is best fit by assuming the cooperative binding of three Co2+ ions with a K0.5 of 39.9 microM, comparable to the value of 16.6 microM obtained kinetically. Displacement of Co2+ by 1 equiv of Zn2+, which binds tightly to the A site of the 3',5'-exonuclease, shifts the optical spectrum to 524 nm and lowers the extinction coefficient to 30 -1 cm-1, indicative of octahedral coordination.2+ the formation of the binuclear complex.

Binding Sites↗
PubMed1991

Nucleotide positions responsible for the processivity of the reaction of exonuclease I with oligodeoxyribonucleotides.

The processive hydrolysis of single-stranded oligodeoxyribonucleotides by exonuclease I from Escherichia coli has been investigated. Oligodeoxyribonucleotides and their analogues, which contain either an abasic site or a methylphosphonate internucleotide linkage, were partially hydrolyzed by exonuclease I. The relative dissociation constant for the enzyme and each oligomeric product was calculated from the concentration of that oligomer found in solution and hence released by the enzyme before complete hydrolysis. The results have led to a characterization of the two oligodeoxyribonucleotide domains that bind to exonuclease I. The first domain, which begins at the reactive 3'-terminal phosphodiester and extends to the 7th nucleoside base, requires both phosphodiester monoanions and base residues for its interaction with the enzyme. The second domain includes phosphodiester monoanions in positions 9-13 from the 3'-terminus but does not require nucleoside bases. Methylphosphonate substitutions indicate that only two or three of these phosphodiesters, in variable positions, must remain anionic in order to obtain full enzyme binding. The residues between the two binding domains do not play a significant role in the enzyme-oligomer interaction.

Base Sequence↗