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PubMed1992

cDNA cloning, sequencing, expression and possible domain structure of human APEX nuclease homologous to Escherichia coli exonuclease III.

cDNA encoding the human homologue of mouse APEX nuclease was isolated from a human bone-marrow cDNA library by screening with cDNA for mouse APEX nuclease. The mouse enzyme has been shown to possess four enzymatic activities, i.e., apurinic/apyrimidinic endonuclease, 3'-5' exonuclease, DNA 3'-phosphatase and DNA 3' repair diesterase activities. The cDNA for human APEX nuclease was 1420 nucleotides long, consisting of a 5' terminal untranslated region of 205 nucleotide long, a coding region of 954 nucleotide long encoding 318 amino acid residues, a 3' terminal untranslated region of 261 nucleotide long, and a poly(A) tail. Determination of the N-terminal amino acid sequence of APEX nuclease purified from HeLa cells showed that the mature enzyme lacks the N-terminal methionine. The amino acid sequence of human APEX nuclease has 94% sequence identity with that of mouse APEX nuclease, and shows significant homologies to those of Escherichia coli exonuclease III and Streptococcus pneumoniae ExoA protein. The coding sequence of human APEX nuclease was cloned into the pUC18 SmaI site in the control frame of the lacZ promoter. The construct was introduced into BW2001 (xth-11, nfo-2) strain and BW9109 (delta xth) strain cells of E. coli. The transformed cells expressed a 36.4 kDa polypeptide (the 317 amino acid sequence of APEX nuclease headed by the N-terminal decapeptide derived from the part of pUC18 sequence), and were less sensitive to methylmethanesulfonate and tert-butyl-hydroperoxide than the parent cells. The N-terminal regions of the constructed protein and APEX nuclease were cleaved frequently during the extraction and purification processes of protein to produce the 31, 33 and 35 kDa C-terminal fragments showing priming activities for DNA polymerase on acid-depurinated DNA and bleomycin-damaged DNA. Formation of such enzymatically active fragments of APEX nuclease may be a cause of heterogeneity of purified preparations of mammalian AP endonucleases. Based on analyses of the deduced amino acid sequence and the active fragments of APEX nuclease, it is suggested that the enzyme is organized into two domains, a 6 kDa N-terminal domain having nuclear location signals and 29 kDa C-terminal, catalytic domain.

Amino Acid Sequence↗
PubMed1996

Construction of single amino acid substitution mutants of cloned Bacillus stearothermophilus DNA polymerase I which lack 5'-->3' exonuclease activity.

Two individual amino acid substitutions were engineered at a selected site in the 5' --> 3' exonuclease domain of the cloned Bacillus stearothermophilus DNA polymerase I gene. These mutations resulted in the expression of enzymes lacking the 5' --> 3' exonuclease activity while maintaining normal polymerizing activity. The mutated and non-mutated enzymes were each constitutively expressed in an Escherichia coli host without the use of an exogenous or inducible promoter, and the mutated enzymes were demonstrated to be equivalent to the subtilisin large fragment of the native holoenzyme in sequencing reactions.

Amino Acids↗
PubMed1993

Requirement of homologous recombination functions for viability of the Escherichia coli cell that lacks RNase HI and exonuclease V activities.

rnhA224 and rnhA339::cat mutants which lack RNase HI activity were found to constitutively express the sfiA::lacZ operon fusion in a recA+ lexA(+)-dependent manner. The sfiA::lacZ expression (indicating SOS induction) in rnhA mutants was increased to higher levels by the introduction of the recD1903 or recB21 mutation. The SOS induction in these cells was further enhanced by nutritional shift up from casamino acid medium to Luria broth. Although the extent by which the recD and recB mutations increased the sfiA expression in rnhA mutants was similar, the rnhA224 recB21 double mutant had plating efficiencies that were 25-fold lower on casamino acid plates and 5 x 10(5)-fold lower on Luria broth plates than the respective plating efficiencies of either rnhA224 recD or rnhA::cat recD double mutants. Whereas the recD mutation inactivates the exonuclease activity of the RecBCD (Exo V) enzyme without reducing the recombination proficiency of the mutant, the recB21 mutation abolishes both the exonuclease activity and recombination capability. Therefore, in the absence of both RNase HI and Exo V activities, homologous recombination functions become crucial for viability, particularly in Luria broth. Introduction of mutations in recA, recJ and recN exacerbated the phenotypes. It is proposed that R-loops which persist due to the lack of RNase HI activity can be removed by two alternative routes of DNA repair: one involving Exo V, Exo I and DNA polymerase I, and the other involving both the RecBCD and RecF pathways of homologous recombination activities. The isolation of RNA polymerase mutants that constitutively express the SOS response at high levels and exhibit remarkable broth-sensitivity lend strong support to the contention that increased amounts of the persisting R-loop in rnhA mutants growing in Luria broth give rise to a stronger SOS response.

DNA Helicases↗
PubMed1984

Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing.

A method is described for the rapid generation and cloning of deletion derivatives well-suited for the sequencing of long stretches of DNA. This method is based on two useful features of exonuclease III: (1) processive digestion at a very uniform rate and (2) failure to initiate digestion at DNA ends with four-base 3'-protrusions. The method was applied to a 4570-bp Drosophila genomic DNA fragment cloned in the single-stranded phage vector M 13mp18. An ordered set of deletion clones was made by first cutting replicative form(RF) DNA with two restriction enzymes in the polylinker region of the vector between the Drosophila DNA and the sequencing primer binding site. One enzyme left a four-base 3'-protrusion that protected the remainder of the vector from exonuclease III attack, allowing unidirectional digestion of the insert sequence from the 5'-protruding end left by the other enzyme. Aliquots were removed at uniform intervals, treated with S1 nuclease, Klenow DNA polymerase, T4 DNA ligase, and then used to transfect competent cells. Most of the resulting clones derived from each aliquot were deleted to a predicted extent with only slight scatter, even for deletions of more than 4 kb. The method permits efficient isolation of clusters of deletion breakpoints within small preselected regions of large DNA segments, allowing nonrandom sequence analysis.

Bacteriophage lambda↗
PubMed1994

Involvement of RecE exonuclease and RecT annealing protein in DNA double-strand break repair by homologous recombination.

We demonstrated that a double-stranded (ds) gap in DNA is repaired by a gene conversion mechanism in an Escherichia coli recBC sbcA23 strain, as predicted by the ds break repair models for homologous recombination. The sbcA mutation is known to induce several gene products encoded on the Rac prophage present in most strains of E. coli K-12. These include exonuclease VIII (Exo VIII), a 5' to 3' exonuclease working from the end of a duplex DNA, and RecT, an annealing protein. We found that a rac- strain (lacking the Rac prophage) cannot support this repair. A plasmid carrying part of the Rac prophage supported highly efficient ds gap repair activity in a rac- strain, but two ExoVIII+ recT- plasmids did not. The recE159 mutation that blocks ds gap repair was found to be recT+, since these ExoVIII+ recT- plasmids complemented the recE159 mutation in repair of ultraviolet light damage. From these observations, we conclude that both ExoVIII and RecT are essential for ds gap repair. We discuss their possible roles in the ds break repair reaction.

Bacterial Proteins↗
PubMed2006

The Apollo 5' exonuclease functions together with TRF2 to protect telomeres from DNA repair.

A major issue in telomere research is to understand how the integrity of chromosome ends is preserved . The human telomeric protein TRF2 coordinates several pathways that prevent checkpoint activation and chromosome fusions. In this work, we identified hSNM1B, here named Apollo, as a novel TRF2-interacting factor. Interestingly, the N-terminal domain of Apollo is closely related to that of Artemis, a factor involved in V(D)J recombination and DNA repair. Both proteins belong to the beta-CASP metallo-beta-lactamase family of DNA caretaker proteins. Apollo appears preferentially localized at telomeres in a TRF2-dependent manner. Reduced levels of Apollo exacerbate the sensitivity of cells to TRF2 inhibition, resulting in severe growth defects and an increased number of telomere-induced DNA-damage foci and telomere fusions. Purified Apollo protein exhibits a 5'-to-3' DNA exonuclease activity. We conclude that Apollo is a novel component of the human telomeric complex and works together with TRF2 to protect chromosome termini from being recognized and processed as DNA damage. These findings unveil a previously undescribed telomere-protection mechanism involving a DNA 5'-to-3' exonuclease.

Animals↗
PubMed2005

The yeast Snm1 protein is a DNA 5'-exonuclease.

Interstrand cross-links (ICL) in DNA arise from bifunctional alkylating agents, including nitrogen mustards, mitomycin C and psoralens. Such adducts prevent normal transcription or replication and are mutagenic. Therefore, cellular mechanisms for removing ICL damage are needed to maintain genome stability. Normal ICL repair requires the action of a number of genes, some specific for such damage. The yeast Snm1 protein is one such protein, but its function has been unknown. Incision for ICL repair is normal in mutants lacking Snm1, so it appears to act after the earliest steps. We have used recombinant SNM1 constructs in an Escherichia coli (E. coli) expression system to demonstrate that the yeast gene encodes a 5'-exonuclease. The exonuclease activity is required for Snm1 to be functional in ICL repair.

Cross-Linking Reagents↗
PubMed2003

AP endonuclease 1 has no biologically significant 3(')-->5(')-exonuclease activity.

The 3(')-->5(')-exonucleolytic activity of human apurinic/apyrimidinic endonuclease 1 (APE1) on mispaired DNA at the 3(')-termini of recessed, nicked or gapped DNA molecules was analyzed and compared with the primary endonucleolytic activity. We found that under reaction conditions optimal for AP endonuclease activity the 3(')-->5(')-exonuclease activity of APE1 manifests only at enzyme concentration elevated by 6-7 orders of magnitude. This activity does not show a preference to mismatched compared to matched DNA structures as well as to nicked or gapped DNA substrates in comparison to recessed ones. Therefore, the 3(')-->5(')-exonuclease activity associated with APE1 can hardly be considered as key mechanism that improves fidelity of DNA repair.

Base Pair Mismatch↗
PubMed1997

Evolution of dnaQ, the gene encoding the editing 3' to 5' exonuclease subunit of DNA polymerase III holoenzyme in Gram-negative bacteria.

The nucleotide sequences of the dnaQ genes from Salmonella typhimurium and Buchnera aphidicola, encoding the epsilon-subunit of the DNA polymerase III holoenzyme, have been determined. The Salmonella typhimurium dnaQ protein consists of 243 amino acid residues with a calculated molecular weight of 27224. The Buchnera aphidicola dnaQ protein contains 233 amino acid residues with a calculated molecular weight of 27170. A multiple sequence alignment of the amino acid sequences of the dnaQ proteins and those of DNA polymerase IIIs from Gram-positive bacteria produced six homologous segments. These homologous segments contain highly conserved amino acid sequence motifs involved in catalytically important metal ion bindings (ligands 1, 2 and 3). However, metal ligand 4 is found to be altered in the 3'-5' exonuclease domain of the family C DNA polymerases and dnaQ proteins in Gram-negative bacteria. From these results, we propose that the last common ancestor of the dnaQ gene of Gram-negative bacteria and the DNA polymerase III gene (pol C gene) of Gram-positive bacteria was a single gene containing both 3'-5' exonuclease and DNA polymerase domains and then the dnaQ gene separated from the polymerase gene in Gram-negative bacteria.

Amino Acid Sequence↗
PubMed2000

Universal PCR amplification of mouse immunoglobulin gene variable regions: the design of degenerate primers and an assessment of the effect of DNA polymerase 3' to 5' exonuclease activity.

Degenerate primers were designed for PCR amplification of unknown mouse immunoglobulin (Ig) light (L) and heavy (H) chain variable (V) genes. Each subgroup of mouse Ig gene sequences [Kabat, E.A., Wu, T. T., Perry, H.H., Gottesman, K.S., Foeller, C., 1991. Sequences of Proteins of Immunological Interest, 5th edn. US Department of Health and Human Services, Public Health Service, NIH.] was analyzed, and highly degenerate primers in the framework one (FR1) region were designed. A single highly degenerate FR1 primer sufficed for the amplification of light chains; for heavy chains, a series of FR1 primers was used. At the same time, we assessed the effect of 3' to 5' exonuclease activity of DNA polymerase on the utilization of these degenerate primers. Using Taq polymerase, which lacks 3' to 5' exonuclease activity, we successfully amplified the Ig VL and VH genes expressed in more than a hundred monoclonal hybridoma cell lines reactive against a phosphonamidate hapten. Sequence analysis of the cloned VL and VH genes, 52 of each, showed that they are derived from multiple germline families (10 of the 17 VL families and 9 of the 14 VH families) as recently defined [Martinez, C., Lefranc, M., 1998. The mouse (Mus musculus) immunoglobulin kappa variable (IGKV) genes and joining (IGKJ) segments. Exp. Clin. Immunogenet. 15, 184.]. The universality of our primers was also demonstrated by successful amplification of other mouse hybridoma cell lines that are specific to different antigens.

Animals↗
PubMed1998

Use of T7 gene 6 exonuclease and phosphorothioated primers for the manipulation of HIV-1 infectious clones.

A method is described for the efficient substitution, deletion or insertion of any desired DNA sequence into any viral infectious clones without the limitation of naturally occurring restriction sites. The technique employs the polymerase chain reaction combined with the resistance of 2'-deoxynucleotides 5'-O-(1-thiotriphosphate) dNTPs [S] bonds (phosphorothiate bonds) to the 5'-3' double strand specific T7 gene 6 exonuclease (T7 Exo) digestion. Primers used to amplify the DNA target regions being manipulated present three phosphorothioate bonds from the fifteenth base at the 5' end. The enzyme activity was shown to be completely inhibited by the presence of more than one phosphorothioate residue at the 5' end of the DNA molecules. When the amplification products are submitted to the exonuclease digestion the hydrolytic T7 Exo activity generates a short single strand DNA tail which contains the nucleotide integrity of the 3' strand. Since the ends of two independently amplified products overlap they can regenerate a stable recombinant structure when further combined in the same reaction tube in the presence of T4 DNA ligase. This new method can be used for manipulating an HIV-1 full-length clone belonging to subtype D replacing the env (gp120) gene for an F subtype sequence.

Bacteriophage T7↗
PubMed2001

Exonuclease activity is required for sequence addition and Cdc13p loading at a de novo telomere.

The Saccharomyces cerevisiae Mre11p/Rad50p/Xrs2p (MRX) complex is evolutionarily conserved and functions in DNA repair and at telomeres [1-3]. In vivo, MRX is required for a 5' --> 3' exonuclease activity that mediates DNA recombination at double-strand breaks (DSBs). Paradoxically, abolition of this exonuclease activity in MRX mutants results in shortened telomeric DNA tracts. To further explore the role of MRX at telomeres, we analyzed MRX mutants in a de novo telomere addition assay in yeast cells [4]. We found that the MRX genes were absolutely required for telomerase-mediated addition in this assay. Furthermore, we found that Cdc13p, a single-stranded telomeric DNA binding protein essential for telomere DNA synthesis and protection [5], was unable to bind to the de novo telomeric DNA substrate in cells lacking Rad50p. Based on the results from this model system, we propose that the MRX complex helps to prepare telomeric DNA for the loading of Cdc13p, which then protects the chromosome from further degradation and recruits telomerase and other DNA replication components to synthesize telomeric DNA.

Cyclin B↗
PubMed2003

The expression of Exonuclease III from E. coli in mitochondria of breast cancer cells diminishes mitochondrial DNA repair capacity and cell survival after oxidative stress.

The ability to sensitize cancer cells to radiation would be highly beneficial for successful cancer treatment. One mode of action for ionizing radiation is the induction of cell death through infliction of extensive oxidative damage to cellular DNA, including mitochondrial DNA (mtDNA). The ability of cells to repair mtDNA and otherwise maintain the integrity of their mitochondria is vital for protection of the cells against oxidative damage. Because efficient repair of oxidative damage in mtDNA may play a crucial role in cancer cell resistance, interference with this repair process could be an effective way to achieve a radiation sensitive phenotype in otherwise resistant cancer cells. Successful repair of DNA is achieved through a precise and highly regulated multistep process. Expression of excessive amounts of one of the repair enzymes may cause an imbalance of the whole repair system and lead to the loss of repair efficiency. To study the effects of changing mtDNA repair capacity on overall cell survival following oxidative stress, we expressed a bacterial repair enzyme, Exonuclease III (ExoIII) containing the mitochondrial targeting signal of manganese superoxide dismutase, in a human malignant breast epithelial cell line, MDA-MB-231. Following transfection, specific exonuclease activity was found in mitochondrial extracts. In order to examine the effects on repair of oxidative damage in mtDNA, cells were exposed to the enzyme xanthine oxidase and its substrate hypoxanthine. mtDNA repair was evaluated using quantitative Southern blot analysis. The results revealed that cells expressing ExoIII in mitochondria are deficient in mtDNA repair when compared with control cells that express ExoIII without MTS. This diminished mtDNA repair capacity rendered MDA-MB-231 cells more sensitive to oxidative damage, which resulted in a decrease in their long-term survival following oxidative stress.

Blotting, Southern↗
PubMed2000

The final step in the formation of 25S rRNA in Saccharomyces cerevisiae is performed by 5'-->3' exonucleases.

The final stage in the formation of the two large subunit rRNA species in Saccharomyces cerevisiae is the removal of internal transcribed spacer 2 (ITS2) from the 27SB precursors. This removal is initiated by endonucleolytic cleavage approximately midway in ITS2. The resulting 7S pre-rRNA, which is easily detectable, is then converted into 5.8S rRNA by the concerted action of a number of 3'-->5' exonucleases, many of which are part of the exosome. So far the complementary precursor to 25S rRNA resulting from the initial cleavage in ITS2 has not been detected and the manner of its conversion into the mature species is unknown. Using various yeast strains that carry different combinations of wild-type and mutant alleles of the major 5'-->3' exonucleases Rat1p and Xrn1p, we now demonstrate the existence of a short-lived 25.5S pre-rRNA whose 5' end is located closely downstream of the previously mapped 3' end of 7S pre-rRNA. The 25.5S pre-rRNA is converted into mature 25S rRNA by rapid exonucleolytic trimming, predominantly carried out by Rat1p. In the absence of Rat1p, however, the removal of the ITS2 sequences from 25.5S pre-rRNA can also be performed by Xrn1p, albeit somewhat less efficiently.

Base Sequence↗
PubMed2000

Rapid and direct sequencing of double-stranded DNA using exonuclease III and MALDI-TOF MS.

Application of MALDI-TOF MS to direct sequencing of dsDNA substrates is demonstrated using a strategy that employs exonuclease III digestion of a target sequence. Experimental conditions for exonuclease III have been optimized for this application, including addition of essential divalent metal ion cofactors. A short cation-exchange column was designed to provide efficient sample cleanup and overcome major problems arising from salt interference.

DNA↗
PubMedDate not supplied

Preparation and nuclease activity of hybrid "metallotris(methylpyridinium)porphyrin oligonucleotide" molecules having a 3'-loop for protection against 3'-exonucleases.

A 5'-GCGAAAGC minihairpin structure was added to the 3'-end of an oligonucleotide substituted at the 5'-end by a manganese cationic porphyrin in order to enhance the 3'-exonuclease resistance of these cleaver-antisense molecules. The influence of this minihairpin on the 3'-exonuclease resistance, the binding affinity to a target ssDNA, and the cleaving efficiency of Mn-cationic porphyrin oligonucleotide conjugates was compared to that of the parent molecule without the 3'-hairpin. The results showed that the 3'-hairpin slightly decreased the binding affinity and consequently the cleaving efficiency of the conjugated molecule toward a target sequence, but the much higher nuclease resistance makes 3'-minihairpin-protected metalloporphyrin oligonucleotides good candidates as reactive antisense oligonucleotides for studies on cells.

Base Sequence↗
PubMed1993

Purification and characterization of a yeast DNA polymerase alpha complex with associated primase, 5'-->3' exonuclease, and DNA-dependent ATPase activities.

We have purified a multimeric form of yeast DNA polymerase alpha with DNA polymerase, primase, 5'-->3' exonuclease, and single-stranded (ss) DNA-dependent ATPase activities to near-homogeneity. The molecular mass of complex was 650 kDa with subunits ranging in sizes from 30 to 180 kDa. The alpha-subunit of the complex could be detected by DNA polymerase alpha antibody. No cross-reactivity of polypeptides within the complex was observed with antibodies directed against polymerase delta or epsilon. The multimeric polymerase alpha could be selectively inhibited by p-n-butylphenyl-dGTP (I50 of approximately 0.2 microM), p-n-butylanilino-dATP (I50 of 1.3 microM), and aphidicolin (I50 of 2.5 micrograms/mL). The complex synthesized RNA primers on various ssDNA templates and rapidly elongated these primers into nascent DNA fragments in the presence of required deoxynucleotides. It has a strong 5'-->3' exonuclease activity. In addition, the complex hydrolyzed both ATP and dATP in a ssDNA-dependent manner. Thus, the multiprotein complex of DNA polymerase alpha had multiple activities (primase, polymerase, and ATPase) which could act concertedly to synthesize primers and elongate the primers to nascent DNA fragments in the lagging strand of the fork.

Adenosine Triphosphatases↗
PubMed1992

A single-stranded DNA exonuclease from Schizosaccharomyces pombe.

We have purified to near homogeneity a DNA exonuclease from meiotic cells of Schizosaccharomyces pombe. The enzyme, designated exonuclease II (ExoII), had an apparent molecular weight of 134,000 and was abundant in the cell. It specifically degraded single-stranded DNA in the 5'----3' direction with an apparent Km for 5' DNA ends of 3.6 x 10(-11) M and produced 5' deoxynucleoside monophosphates. Its mode of degradation is similar to that of the RecJ protein from Escherichia coli; ExoII may, therefore, be involved in genetic recombination and DNA damage repair.

Chromatography, Affinity↗