Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Exonucleases”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 55 records · Page 3Linked to original sources
PubMed1989

Mechanism of action of Escherichia coli exonuclease III.

Exonuclease III is the major apurinic/apyrimidinic (AP) endonuclease of Escherichia coli, accounting for more than 80% of the total cellular AP endonuclease activity. We have shown earlier that the endonucleolytic activity of exonuclease III is able to hydrolyze the phosphodiester bond 5' to the urea N-glycoside in a duplex DNA [Kow, Y. W., & Wallace, S. S. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 8354-8358]. Therefore, we were interested in studying the mechanism of action of the endonucleolytic activity of exonuclease III by preparing DNA containing different base lesions as well as chemically modified AP sites. When AP sites were converted to O-alkylhydroxylamine residues, exonuclease III was able to hydrolyze the phosphodiester bond 5' to O-alkylhydroxylamine residues. The apparent Km for different O-alkylhydroxylamine residues was not affected by the particular O-alkylhydroxylamine residue substituted; however, the apparent Vmax decreased as the size of the residue increased. On the basis of a study of the substrate specificity of exonuclease III, a modification of the Weiss model for the mechanism of action of exonuclease III is presented. Furthermore, a temperature study of exonucleolytic activity of exonuclease III in the presence of Mg2+ showed discontinuity in the Arrhenius plot. However, no discontinuity was observed when the reaction was performed in the presence of Ca2+. Similarly, no discontinuity was observed for the endonucleolytic activity of exonuclease III, in the presence of either Ca2+ or Mg2+. These data suggest that, in the presence of Mg2+, exonuclease III, in the presence of either Ca2+ or Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1998

Effects of mutations on the partitioning of DNA substrates between the polymerase and 3'-5' exonuclease sites of DNA polymerase I (Klenow fragment).

Site-directed mutagenesis and time-resolved fluorescence spectroscopy were used to evaluate the contributions of individual amino acid side chains to the binding of DNA primer-templates to the 3'-5' exonuclease site of the large proteolytic fragment (Klenow fragment) of DNA polymerase I. Mutations were introduced into side chains that have been shown crystallographically to be in close proximity to a DNA 3' terminus bound at the 3'-5' exonuclease site. The wild-type residues were replaced by alanine in each case. To assess the effects of the mutations on DNA binding, time-resolved fluorescence anisotropy measurements were performed on dansyl-labeled primer-templates bound to the mutant enzymes. In contrast to techniques that simply monitor the overall binding of proteins to DNA, the time-resolved fluorescence anisotropy technique was used to determine the fractional occupancies of the polymerase and 3'-5' exonuclease active sites of Klenow fragment. Equilibrium constants describing the partitioning of DNA between the two active sites were obtained for nine different mutant enzymes bound to both matched and mismatched DNA sequences. Mutations of Leu361 and Phe473 caused the largest effects, significantly destabilizing the binding of mismatched DNA substrates to the 3'-5' exonuclease site relative to DNA bound at the polymerase site, consistent with structural data showing that the side chains of these residues are involved in intimate hydrophobic interactions with the 3' terminal and penultimate bases of the primer strand [Beese, L., and Steitz, T. A. (1991) EMBO J. 10, 25-33]. Mutations of the His660 and Glu357 side chains also resulted in significant effects on the binding of mismatched DNA to the 3'-5' exonuclease site. Surprisingly, mutation of Tyr497 increased the partitioning of mismatched DNA into the 3'-5' exonuclease site, suggesting that the tyrosine side chain in the wildtype enzyme destabilizes substrate binding, despite crystallographic data showing that Tyr497 is H-bonded to the DNA substrate. The effects of mutating the amino acid side chains that serve as ligands to two divalent metal ions bound at the 3'-5' exonuclease site, designated A and B, indicated that metal A also helps to bind DNA to the 3'-5' exonuclease site. These results demonstrate that the time-resolved fluorescence anisotropy technique can be used to quantify the energetic contributions associated with each of the crystallographically defined DNA-protein contacts at the 3'-5' exonuclease site.

Amino Acid Substitution↗
PubMed2002

Characterization of an endonuclease IV 3'-5' exonuclease activity.

Previous characterization of Escherichia coli endonuclease IV has shown that the enzyme specifically cleaves the DNA backbone at apurinic/apyrimidinic sites and removes 3' DNA blocking groups. By contrast, and unlike the major apurinic/apyrimidinic endonuclease exonuclease III, negligible exonuclease activity has been associated with endonuclease IV. Here we report that endonuclease IV does possess an intrinsic 3'-5' exonuclease activity. The activity was detected in purified preparations of the endonuclease IV protein from E. coli and from the distantly related thermophile Thermotoga maritima; it co-eluted with both enzymes under different chromatographic conditions. Induction of either endonuclease IV in an E. coli overexpression system resulted in induction of the exonuclease activity, and the E. coli exonuclease activity had similar heat stability to the endonuclease IV AP endonuclease activity. Characterization of the exonuclease activity showed that its progression on substrate is sensitive to ionic strength, metal ions, EDTA, and reducing conditions. Substrates with 3' recessed ends were preferred substrates for the 3'-5' exonuclease activity. Comparison of the relative apurinic/apyrimidinic endonuclease and exonuclease activity of endonuclease IV shows that the relative exonuclease activity is high and is likely to be significant in vivo.

Cations, Divalent↗
PubMed1998

Single-strand DNA-specific exonucleases in Escherichia coli. Roles in repair and mutation avoidance.

Mutations in the genes encoding single-strand DNA-specific exonucleases (ssExos) of Escherichia coli were examined for effects on mutation avoidance, UV repair, and conjugational recombination. Our results indicate complex and partially redundant roles for ssExos in these processes. Although biochemical experiments have implicated RecJ exonuclease, Exonuclease I (ExoI), and Exonuclease VII (ExoVII) in the methyl-directed mismatch repair pathway, the RecJ- ExoI- ExoVII- mutant did not exhibit a mutator phenotype in several assays for base substitution mutations. If these exonucleases do participate in mismatch excision, other exonucleases in E. coli can compensate for their loss. Frameshift mutations, however, were stimulated in the RecJ- ExoI- ExoVII- mutant. For acridine-induced frameshifts, this mutator effect was due to a synergistic effect of ExoI- and ExoVII- mutations, implicating both ExoI and ExoVII in avoidance of frameshift mutations. Although no single exonuclease mutant was especially sensitive to UV irradiation, the RecJ- ExoVII- double mutant was extremely sensitive. The addition of an ExoI- mutation augmented this sensitivity, suggesting that all three exonucleases play partially redundant roles in DNA repair. The ability to inherit genetic markers by conjugation was reduced modestly in the ExoI- RecJ- mutant, implying that the function of either ExoI or RecJ exonucleases enhances RecBCD-dependent homologous recombination.

Bacterial Proteins↗
PubMed1994

Identification of a 3'-->5'-exonuclease that removes cytosine arabinoside monophosphate from 3' termini of DNA.

Cytosine arabinoside monophosphate (araCMP) at the 3' terminus of DNA constitutes a lesion that impedes further synthesis by DNA polymerase alpha (DNA pol alpha). A biochemical assay has been designed to detect 3'-->5'-exonucleases in cell extracts that remove the 3'-araCMP lesion in an oligonucleotide template-primer and permit subsequent extension by DNA pol alpha. The major 3'-->5'-exonuclease activity in human myeloblast extracts has been purified, and gel filtration chromatography of the purified enzyme indicates that the exonuclease has an apparent native molecular mass of 52 kDa. Incubation of the enzyme with a 5'-32P-labeled araCMP template-primer results in exonucleolytic degradation of the primer exclusively in the 3'-->5' direction, demonstrating that the enzyme is a 3'-->5'-exonuclease. The products of the 3'-->5'-exonuclease reaction are 5'-mononucleotides. The apparent rate of araCMP removal by the exonuclease is approximately the same as the rate of deoxynucleoside monophosphate (dNMP) removal. Furthermore, the apparent rates of 3'-terminal excision are approximately the same whether the oligomer is hybridized to a complementary oligonucleotide, or not, indicating that the enzyme has both single- and double-stranded 3'-->5'-exonuclease activity. The enzyme does not possess 5'-->3'-exonuclease activity, nor is it associated with DNA polymerase activity. In addition, the enzyme does not cleave 3'-phosphoryl-terminated DNA, and it does not cleave RNA. The enzymatic characteristics of the isolated 3'-->5'-exonuclease indicate that it is distinct from previously identified mammalian deoxyribonucleases.

Arabinonucleotides↗
PubMed1990

Role of gene 6 exonuclease in the replication and packaging of bacteriophage T7 DNA.

When bacteriophage T7 gene 6 exonuclease is genetically removed from T7-infected cells, degradation of intracellular T7 DNA is observed. By use of rate zonal centrifugation, followed by either pulsed-field agarose gel electrophoresis or restriction endonuclease analysis, in the present study, the following observations were made. (1) Most degradation of intracellular DNA requires the presence of T7 gene 3 endonuclease and is independent of DNA packaging; rapidly sedimenting, branched DNA accumulates when both the gene 3 and gene 6 products are absent. (2) A comparatively small amount of degradation requires packaging and occurs at both the joint between genomes in a concatemer and near the left end of intracellular DNA; DNA packaging is only partially blocked and end-to-end joining of genomes is not blocked in the absence of gene 6 exonuclease. (3) Fragments produced in the absence of gene 6 exonuclease are linear and do not further degrade; precursors of the fragments are non-linear. (4) Some, but not most, of the cleavages that produce these fragments occur selectively near two known origins of DNA replication. On the basis of these observations, the conclusion is drawn that most degradation that occurs in the absence of T7 gene 6 exonuclease is caused by cleavage at branches. The following hypothesis is presented: most, possibly all, of the extra branching induced by removal of gene 6 exonuclease is caused by strand displacement DNA synthesis at the site of RNA primers of DNA synthesis; the RNA primers, produced by multiple initiations of DNA replication, are removed by the RNase H activity of gene 6 exonuclease during a wild-type T7 infection. Observation of joining of genomes in the absence of gene 6 exonuclease and additional observations indicate that single-stranded terminal repeats required for concatamerization are produced by DNA replication. The observed selective shortening of the left end indicates that gene 6 exonuclease is required for formation of most, possibly all, mature left ends.

Centrifugation, Density Gradient↗
PubMed2005

Contribution of polar residues of the J-helix in the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I (Klenow fragment): Q677 regulates the removal of terminal mismatch.

Previous structural and biochemical data indicate a participation of the J-helix of Escherichia coli pol I in primer positioning at the polymerase and exonuclease sites. The J-helix contains three polar residues: N675, Q677, and N678. Preliminary characterization of alanine substitutions of these residues showed that only Q677A DNA polymerase has substantially decreased polymerase and increased exonuclease activity. The Q677A enzyme had approximately 2- and approximately 5-fold greater exonuclease activity than the wild type (WT) with mismatched and matched template-primers (TPs), respectively. N675A and N678A DNA polymerases did not differ significantly from the WT in these activities, despite the fact that both residues are seen to interact with the TP in various pol I-DNA complexes. Pre-steady-state kinetic measurements for the exonuclease activity of WT and mutant enzymes indicated nearly identical DNA binding affinity for ssDNA and mismatched TPs. However, with a matched TP, Q677A DNA polymerase exhibited increased exonuclease site affinity. The most important characteristic of Q677A DNA polymerase was its ability to continue cleavage into the matched region of the TP after mismatch excision, in contrast to the WT and other mutant enzymes. The increase in the exonuclease activity of Q677A DNA polymerase was further determined not to be solely due to the weakened binding at the polymerase site, by comparison with another polymerase-defective mutant enzyme, namely, R668A DNA polymerase. These enzymes have significantly decreased DNA binding affinity at the polymerase site, yet the exonuclease activity parameters of R668A DNA polymerase remain similar to those of the WT. These results strongly suggest that participation of Q677 is required for positioning the primer terminus (a) in the polymerase site for continued nucleotide addition and (b) in the 3'-exonuclease site for the controlled removal of mismatched nucleotides.

Alanine↗
PubMed1998

Exonuclease-polymerase active site partitioning of primer-template DNA strands and equilibrium Mg2+ binding properties of bacteriophage T4 DNA polymerase.

The binding of bacteriophage T4 DNA polymerase (T4 pol) to primer-template DNA with 2-aminopurine (2AP) located at the primer terminus results in the formation of a hyperfluorescent 2AP state. Changes in this hyperfluorescent state were utilized to investigate the fractional concentration of primer-templates bound at the exonuclease and statically quenched polymerase sites. In the absence of Mg2+, a hydrophobic exonuclease site dominates over the polymerase site for possession of the primer terminus. The fractional concentration of primer termini in the exonuclease site was found to be 64 and 84% for correct (AP-T) and mismatched (AP-C) primer-templates, respectively. Exonuclease-deficient mutants, polymerase-switching mutants, and nucleoside triphosphates all shift this equilibrium toward the polymerase site. Synthesis of stereospecific hydrolysis resistant phosphorothioate 2AP-labeled DNA allowed Mg2+ ion binding titrations to be performed in the presence of bound DNA without the complication of the excision reaction. High- and low-affinity Mg2+ binding sites were observed in the presence of bound double-stranded (ds) DNA, with dissociation constants in the micromolar (WT Kd = 5.1 microM) and millimolar (WT Kd = 2.5 mM) concentration ranges. Mg2+ binding was found to be a key "conformational switch" for T4 pol. As the high-affinity Mg2+ binding sites are filled, the primer terminus migrates from the exonuclease site to a highly based stacked polymerase active site. Filling the low-affinity Mg2+ sites further shifts the primer terminus into the polymerase site. As the low-affinity Mg2+ sites are filled, T4 pol "loosens its grip" on the primer terminus, as shown by a large amplitude increase in the nanosecond rotational mobility of 2AP within the bound T4 complex. The hyperfluorescent exonuclease site is spatially localized to 2AP positioned on the primer end. The penultimate (n - 1) position, as well as n - 2 and n - 5 positions, reveals no detectable fluorescent enhancement upon binding. The observed position-dependent fluorescence data, when combined with time-resolved total-intensity and anisotropy data, suggest that the creation of the hyperfluorescent state is caused by phenylalanine 120 (F120) of T4 pol intercalating into 2AP primers much like that observed for phenylalanine 123 of RB69 DNA polymerase intercalating into deoxythymidine primers [Wang, J., et al. (1997) Cell 89, 1087-1099]. As Mg2+ binds in the exonuclease site of T4 pol, the primer terminus appears to be "pulled backward" into the active site, decreasing the concentration of F120-intercalated primer termini, and bringing the exonuclease active site residues closer to the primer terminus scissile phosphate bond.

2-Aminopurine↗
PubMed2005

A conserved and species-specific functional interaction between the Werner syndrome-like exonuclease atWEX and the Ku heterodimer in Arabidopsis.

Werner syndrome is associated with mutations in the DNA helicase RecQ3 [a.k.a. Homo sapiens (hs)WRN]. The function of hsWRN is unknown although biochemical studies suggest a role in DNA ends stability and repair. Unlike other RecQ family members, hsWRN possesses an N-terminal domain with exonuclease activity, which is stimulated by interaction with the Ku heterodimer. While this interaction is intriguing, we do not know whether it is important for hsWRN function. Although flies, worms, fungi and plants do not have RecQ-like (RQL) helicases with an intrinsic exonuclease activity, they possess proteins having domains homologous to the hsWRN exonuclease. The genome of Arabidopsis thaliana (at) encodes multiple RQL and a single protein with homology to the WRN exonuclease domain, atWEX (Werner-like Exonuclease). Here we show that atWEX has properties that are similar to hsWRN. atWEX binds to and is stimulated by atKu. Interestingly, stimulation by Ku is species-specific, as hsKu does not stimulate atWEX exonuclease activity. Likewise, atKu fails to enhance the exonuclease activity of hsWRN. Thus, in spite of the differences in structural organization, the functional interaction between WRN-like exonucleases and Ku has been preserved through evolutionary radiation of species, emphasizing the importance of this interaction in cell function.

Amino Acid Sequence↗
PubMed2004

The DNA double-stranded break repair protein endo-exonuclease as a therapeutic target for cancer.

DNA repair mechanisms are crucial for the maintenance of genomic stability and are emerging as potential therapeutic targets for cancer. In this study, we report that the endo-exonuclease, a protein involved in the recombination repair process of the DNA double-stranded break pathway, is overexpressed in a variety of cancer cells and could represent an effective target for developing anticancer drugs. We identify a dicationic diarylfuran, pentamidine, which has been used clinically to treat opportunistic infections and is an inhibitor of the endo-exonuclease as determined by enzyme kinetic assay. In clonogenic and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays as well as in the in vivo Lewis lung carcinoma mouse tumor model, pentamidine is shown to possess the ability to selectively kill cancer cells. The LD50 of pentamidine on cancer cells maintained in vitro is correlated with the endo-exonuclease enzyme activity. Tumor cell that has been treated with pentamidine is reduced in the endo-exonuclease as compared with the untreated control. Furthermore, pentamidine synergistically potentiates the cytotoxic effect of DNA strand break and cross-link-inducing agents such as mitomycin C, etoposide, and cisplatin. In addition, we used the small interfering RNA for the mouse homologue of the endo-exonuclease to down-regulate the level of endo-exonuclease in the mouse myeloma cell line B16F10. Down-regulation of the endo-exonuclease sensitizes the cell to 5-fluorouracil. These studies suggested the endo-exonuclease enzyme as a novel potential therapeutic target for cancer.

Animals↗
PubMed1991

Properties of the 3' to 5' exonuclease associated with porcine liver DNA polymerase gamma. Substrate specificity, product analysis, inhibition, and kinetics of terminal excision.

Porcine liver DNA polymerase gamma was shown previously to copurify with an associated 3' to 5' exonuclease activity (Kunkel, T. A., and Mosbaugh, D. W. (1989) Biochemistry 28, 988-995). The 3' to 5' exonuclease has now been characterized, and like the DNA polymerase activity, it has an absolute requirement for a divalent metal cation (Mg2+ or Mn2+), a relatively high NaCl and KCl optimum (150-200 mM), and an alkaline pH optimum between 7 and 10. The exonuclease has a 7.5-fold preference for single-stranded over double-stranded DNA, but it cannot excise 3'-terminal dideoxy-NMP residues from either substrate. Excision of 3'-terminally mismatched nucleotides was preferred approximately 5-fold over matched 3' termini, and the hydrolysis product from both was a deoxyribonucleoside 5'-monophosphate. The kinetics of 3'-terminal excision were measured at a single site on M13mp2 DNA for each of the 16 possible matched and mismatched primer.template combinations. As defined by the substrate specificity constant (Vmax/Km), each of the 12 mismatched substrates was preferred over the four matched substrates (A.T, T.A, C.G, G.C). Furthermore, the exonuclease could efficiently excise internally mismatched nucleotides up to 4 residues from the 3' end. DNA polymerase gamma was not found to possess detectable DNA primase, endonuclease, 5' to 3' exonuclease, RNase, or RNase H activities. The DNA polymerase and exonuclease activities exhibited dissimilar rates of heat inactivation and sensitivity to N-ethylmaleimide. After nondenaturing activity gel electrophoresis, the DNA polymerase and 3' to 5' exonuclease activities were partially resolved and detected in situ as separate species. A similar analysis on a denaturing activity gel identified catalytic polypeptides with molecular weights of 127,000, 60,000, and 32,000 which possessed only DNA polymerase gamma activity. Collectively, these results suggest that the polymerase and exonuclease activities reside in separate polypeptides, which could be derived from separate gene products or from proteolysis of a single gene product.

Animals↗
PubMed1978

Processivity of DNA exonucleases.

A homopolymer system has been developed to examine the digestion strategies of DNA exonucleases. Escherichia coli exonuclease I and lambda-exonuclease, are processive enzymes. However, T7 exonuclease, spleen exonuclease, E. coli exonuclease III, the 3' leads to 5'-exonuclease of T4 DNA polymerase, and both the 3' leads to 5' and the 5' leads to 3' activity of E. coli DNA polymerase I dissociate frequently from the substrate during the course of digestion. Regions of duplex DNA are a dissociation signal for exonuclease I.

Animals↗
PubMed2002

3'-5' exonuclease of Klenow fragment: role of amino acid residues within the single-stranded DNA binding region in exonucleolysis and duplex DNA melting.

The mechanism of the 3'-5' exonuclease activity of the Klenow fragment of DNA polymerase I has been investigated with a combination of biochemical and spectroscopic techniques. Site-directed mutagenesis was used to make alanine substitutions of side chains that interact with the DNA substrate on the 5' side of the scissile phosphodiester bond. Kinetic parameters for 3'-5' exonuclease cleavage of single- and double-stranded DNA substrates were determined for each mutant protein in order to probe the role of the selected side chains in the exonuclease reaction. The results indicate that side chains that interact with the penultimate nucleotide (Q419, N420, and Y423) are important for anchoring the DNA substrate at the active site or ensuring proper geometry of the scissile phosphate. In contrast, side chains that interact with the third nucleotide from the DNA terminus (K422 and R455) do not participate directly in exonuclease cleavage of single-stranded DNA. Alanine substitutions of Q419, Y423, and R455 have markedly different effects on the cleavage of single- and double-stranded DNA, causing a much greater loss of activity in the case of a duplex substrate. Time-resolved fluorescence anisotropy decay measurements with a dansyl-labeled primer/template indicate that the Q419A, Y423A, and R455A mutations disrupted the ability of the Klenow fragment to melt duplex DNA and bind the frayed terminus at the exonuclease site. In contrast, the N420A mutation stabilized binding of a duplex terminus to the exonuclease site, suggesting that the N420 side chain facilitates the 3'-5' exonuclease reaction by introducing strain into the bound DNA substrate. Together, these results demonstrate that protein side chains that interact with the second or third nucleotides from the terminus can participate in both the chemical step of the exonuclease reaction, by anchoring the substrate in the active site or by ensuring proper geometry of the scissile phosphate, and in the prechemical steps of double-stranded DNA hydrolysis, by facilitating duplex melting.

Amino Acids↗
PubMed2003

Stabilization of perfect and imperfect tandem repeats by single-strand DNA exonucleases.

Rearrangements between tandemly repeated DNA sequences are a common source of genetic instability. Such rearrangements underlie several human genetic diseases. In many organisms, the mismatch-repair (MMR) system functions to stabilize repeats when the repeat unit is short or when sequence imperfections are present between the repeats. We show here that the action of single-stranded DNA (ssDNA) exonucleases plays an additional, important role in stabilizing tandem repeats, independent of their role in MMR. For perfect repeats of approximately 100 bp in Escherichia coli that are not susceptible to MMR, exonuclease (Exo)-I, ExoX, and RecJ exonuclease redundantly inhibit deletion. Our data suggest that >90% of potential deletion events are avoided by the combined action of these three exonucleases. Imperfect tandem repeats, less prone to rearrangements, are stabilized by both the MMR-pathway and ssDNA-specific exonucleases. For 100-bp repeats containing four mispairs, ExoI alone aborts most deletion events, even in the presence of a functional MMR system. By genetic analysis, we show that the inhibitory effect of ssDNA exonucleases on deletion formation is independent of the MutS and UvrD proteins. Exonuclease degradation of DNA displaced during the deletion process may abort slipped misalignment. Exonuclease action is therefore a significant force in genetic stabilization of many forms of repetitive DNA.

Base Pair Mismatch↗
PubMed1999

Identification and expression of the TREX1 and TREX2 cDNA sequences encoding mammalian 3'-->5' exonucleases.

The 3'-->5' exonucleases catalyze the excision of nucleoside monophosphates from the 3' termini of DNA. We have identified the cDNA sequences encoding two 3'-->5' exonucleases (TREX1 and TREX2) from mammalian cells. The TREX1 and TREX2 proteins are 304 and 236 amino acids in length, respectively. Analysis of the TREX1 and TREX2 sequences identifies three conserved motifs that likely generate the exonuclease active site in these enzymes. The specific amino acids in these three conserved motifs suggest that these mammalian exonucleases are most closely related to the proofreading exonucleases of the bacterial replicative DNA polymerases and the RNase T enzymes. Expression of TREX1 and TREX2 in Escherichia coli demonstrates that these recombinant proteins are active 3'-->5' exonucleases. The recombinant TREX1 protein was purified, and exonuclease activity was measured using single-stranded, partial duplex, and mispaired oligonucleotide DNA substrates. The greatest activity of the TREX1 protein was detected using a partial duplex DNA containing five mispaired nucleotides at the 3' terminus. No activity was detected using single-stranded RNA or an RNA-DNA partial duplex. Identification of the TREX1 and TREX2 cDNA sequences provides the genetic tools to investigate the physiological roles of these exonucleases in mammalian DNA replication, repair, and recombination pathways.

Amino Acid Sequence↗
PubMed2001

The 3' --> 5' exonuclease of T4 DNA polymerase removes premutagenic alkyl mispairs and contributes to futile cycling at O6-methylguanine lesions.

We have studied the processing of O(6)-methylguanine (m6G)-containing oligonucleotides and N-methyl-N-nitrosourea (MNU)-treated DNA templates by the 3' --> 5' exonuclease of T4 DNA polymerase. In vitro biochemical analyses demonstrate that the exonuclease can remove bases opposite a defined m6G lesion. The efficiency of excision of a terminal m6G.T was similar to that of m6G.C, and both were excised as efficiently as a G.T substrate. Partitioning assays between the polymerase and exonuclease activities, performed in the presence of dNTPs, resulted in repeated incorporation and excision events opposite the m6G lesion. This idling produces dramatically less full-length product, relative to natural substrates, indicating that the 3' --> 5' exonuclease may contribute to DNA synthesis inhibition by alkylating agents. Genetic data obtained using an in vitro herpes simplex virus-thymidine kinase assay support the inefficiency of the exonuclease as a "proofreading" activity for m6G, since virtually all mutations produced by the native enzyme using MNU-treated templates were G --> A transitions. Comparison of MNU dose-response curves for exonuclease-proficient and -deficient forms of T4 polymerase reveals that the exonuclease efficiently removes 50-86% of total premutagenic alkyl mispairs. We propose that idling of exonuclease-proficient polymerases at m6G lesions during repair DNA synthesis provides the biochemical explanation for cellular cytotoxicity of methylating agents.

Alkylating Agents↗
PubMed1996

Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity.

A thermostable DNA polymerase, the Bst DNA polymerase I, from Bacillus stearothermophilus N3468 was prepared to near-homogeneity. The dominant species of the Bst DNA polymerase I preparation sized about 97 kDa when analyzed on SDS polyacrylamide gels. The Bst polA gene that codes for Bst polymerase I was cloned and sequenced. Comparative sequence analysis showed that all three conserved 3'-->5' exonuclease motifs found in E. coli DNA polymerase I were missing in Bst DNA polymerase I. This cast doubt on the existence of a 3'-->5' exonuclease function in that enzyme. Four biochemical assays were used to measure exonuclease activities of Bst DNA polymerase I, testing both full-length Bst polymerase I and the Bst large fragment which lacks the N-terminal 5'-->3' exonuclease domain. These exonuclease assays demonstrated that Bst DNA polymerase I only contained a double-strand dependent 5'-->3' exonuclease activity but lacked any detectable 3'-->5' proofreading exonuclease activity. The lack of 3'-->5' exonuclease function in a variety of thermostable repair DNA polymerases may reflect enhancement of thermostability at the expense of proofreading activity.

Amino Acid Sequence↗
PubMed1994

Phosphatase activity in commercial spleen exonuclease decreases the recovery of benzo[a]pyrene and N-hydroxy-2-naphthylamine DNA adducts by 32P-postlabeling.

Spleen exonuclease, which degrades nucleic acids into single 3'-nucleotides, is used in the detection of DNA adducts by 32P-postlabeling. Contamination of the exonuclease with phosphatase activity can reduce the recovery of benzo[a]pyrene and N-hydroxy-2-naphthylamine DNA adducts by 32P-postlabeling. Four preparations of spleen exonuclease containing varying levels of phosphatase activity (< 1-62% of the unmodified 3'-nucleotides being dephosphorylated) were used to hydrolyze the DNA. The exonuclease with the lowest phosphatase activity produced a recovery of up to 9.60 mumol of benzo[a]pyrene adducts per mole of DNA. Recovery of benzo[a]pyrene adducts was reduced to 0.56 mumol of adduct per mole of DNA using the exonuclease with the highest phosphatase activity. Phosphatase in the exonucleases also dephosphorylated N-hydroxy-2-naphthylamine DNA adducts. Surprisingly, recovery of these DNA adducts was nearly 10 times greater using nuclease P1 than when using 1-butanol extraction for adduct enrichment, since arylamine DNA adducts have previously been reported to be poorly detected by 32P-postlabeling after nuclease P1 treatment. Our data indicate that the hydrolysis of DNA by spleen exonuclease may be an important source of variability in both qualitative and quantitative analysis of adducts by 32P-postlabeling.

2-Naphthylamine↗