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 739 records · Page 41Linked to original sources
PubMed1996

Trypanosome U-deletional RNA editing involves guide RNA-directed endonuclease cleavage, terminal U exonuclease, and RNA ligase activities.

We have studied the mechanism of accurate in vitro RNA editing of Trypanosoma brucei ATPase 6 mRNA, using four mRNA-guide RNA (gRNA) pairs that specify deletion of 2, 3, or 4 U residues at editing site 1 and mitochondrial extract. This extract not only catalyzes deletion of the specified number of U residues but also exhibits a novel endonuclease activity that cleaves the input pre-mRNA in a gRNA-directed manner, precisely at the phosphodiester bond predicted in a simple enzymatic model of RNA editing. This cleavage site is inconsistent with a chimera-based editing mechanism. The U residues to be deleted, present at the 3' end of the upstream cleavage product, are then removed evidently by a 3' U-specific exonuclease and not by a reverse reaction of terminal U transferase. RNA ligase can then join the mRNA halves through their newly formed 5' P and 3' OH termini, generating mRNA faithfully edited at the first editing site. This resultant, partially edited mRNA can then undergo accurate, gRNA-directed cleavage at editing site 2, again precisely as predicted by the enzymatic editing model. All of these enzymatic activities cofractionate with the U-deletion activity and may reside in a single complex. The data imply that each round of editing is a four-step process, involving (i) gRNA-directed cleavage of the pre-mRNA at the bond immediately 5' of the region base paired to the gRNA, (ii) U deletion from or U addition to the 3' OH of the upstream mRNA half, (iii) ligation of the mRNA halves, and (iv) formation of additional base pairing between the correctly edited site and the gRNA that directs subsequent nuclease cleavage at the next editing site.

Animals↗
PubMed1998

Efficient translesion replication in the absence of Escherichia coli Umu proteins and 3'-5' exonuclease proofreading function.

Translesion replication (TR) past a cyclobutane pyrimidine dimer in Escherichia coli normally requires the UmuD'2C complex, RecA protein, and DNA polymerase III holoenzyme (pol III). However, we find that efficient TR can occur in the absence of the Umu proteins if the 3'-5' exonuclease proofreading activity of the pol III epsilon-subunit also is disabled. TR was measured in isogenic uvrA6 DeltaumuDC strains carrying the dominant negative dnaQ allele, mutD5, or DeltadnaQ spq-2 mutations by transfecting them with single-stranded M13-based vectors containing a specifically located cis-syn T-T dimer. As expected, little TR was observed in the DeltaumuDC dnaQ+ strain. Surprisingly, 26% TR occurred in UV-irradiated DeltaumuDC mutD5 cells, one-half the frequency found in a uvrA6 umuDC+mutD5 strain. lexA3 (Ind-) derivatives of the strains showed that this TR was contingent on two inducible functions, one LexA-dependent, responsible for approximately 70% of the TR, and another LexA-independent, responsible for the remaining approximately 30%. Curiously, the DeltaumuDC DeltadnaQ spq-2 strain exhibited only the LexA-independent level of TR. The cause of this result appears to be the spq-2 allele, a dnaE mutation required for viability in DeltadnaQ strains, since introduction of spq-2 into the DeltaumuDC mutD5 strain also reduces the frequency of TR to the LexA-independent level. The molecular mechanism responsible for the LexA-independent TR is unknown but may be related to the UVM phenomenon [Palejwala, V. A., Wang, G. E., Murphy, H. S. & Humayun, M. Z. (1995) J. Bacteriol. 177, 6041-6048]. LexA-dependent TR does not result from the induction of pol II, since TR in the DeltaumuDC mutD5 strain is unchanged by introduction of a DeltapolB mutation.

Alleles↗
PubMed2000

Human DNA damage checkpoint protein hRAD9 is a 3' to 5' exonuclease.

Human RAD9 protein (hRAD9) is a homolog of the fission yeast Rad9 protein, one of the six so-called checkpoint Rad proteins involved in the early steps of DNA damage checkpoint response in Schizosaccharomyces pombe. It has been shown previously that, in vivo, a highly modified form of hRAD9 makes a ternary complex with two other checkpoint Rad proteins, hRAD1 and hHUS1 (Volkmer, E., and Karnitz, L. M. (1999) J. Biol. Chem. 274, 567-570; St. Onge, R. P., Udell, C. M., Casselman, R., and Davey, S. (1999) Mol. Biol. Cell. 10, 1985-1995). However, the function of this complex is not known at present. To help define the functions of checkpoint Rad proteins in humans, we expressed hRAD9 in Escherichia coli, purified the recombinant protein and characterized it. We found that hRAD9 is a 3' to 5' exonuclease and located the nuclease active site to the region between residues 51 and 91 of the 391-amino acid-long protein. Our results suggest that exonucleolytic processing of primary DNA lesion by hRAD9 may contribute to DNA damage checkpoint response in humans.

Cell Cycle Proteins↗
PubMed2001

Structure and expression of the TREX1 and TREX2 3' --> 5' exonuclease genes.

The TREX1 and TREX2 genes encode mammalian 3'-->5' exonucleases. Expression of the TREX genes in human cells was investigated using a reverse transcription-polymerase chain reaction strategy. Our results show that TREX1 and TREX2 are expressed in all tissues tested, providing direct evidence for the expression of these genes in human cells. Potential transcription start sites are identified for the TREX genes using rapid amplification of cDNA ends to recover the 5'-flanking regions of the TREX transcripts. The 5'-flanking sequences indicate transcription initiation from consensus putative promoters identified -140 and -650 base pairs upstream of the TREX1 open reading frame (ORF) and -623 and -753 base pairs upstream of the TREX2 ORF. Novel TREX1 and TREX2 cDNAs are identified that contain protein-coding sequences generated from exons positioned in genomic DNA up to 18 kilobases 5' to the TREX1 ORF and up to 25 kilobases 5' to the TREX2 ORF. These novel cDNAs and sequences in the GenBank data base indicate that transcripts containing the TREX1 and TREX2 ORFs are produced using a variety of mechanisms that include alternate promoter usage, alternative splicing, and varied sites for 3' cleavage and polyadenylation. These initial studies have revealed previously unrecognized complexities in the structure and expression of the TREX1 and TREX2 genes.

Amino Acid Sequence↗
PubMed2004

Analysis of the effect of bulk at N2-alkylguanine DNA adducts on catalytic efficiency and fidelity of the processive DNA polymerases bacteriophage T7 exonuclease- and HIV-1 reverse transcriptase.

The N-2 atom of guanine (G) is susceptible to modification by various carcinogens. Oligonucleotides with increasing bulk at this position were analyzed for fidelity and catalytic efficiency with the processive DNA polymerases human immunodeficiency virus, type 1, reverse transcriptase (RT), and bacteriophage T7 exonuclease(-) (T7(-)). RT and T7(-) effectively bypassed N(2)-methyl(Me)G and readily extended primers but were strongly blocked by N(2)-ethyl(Et)G, N(2)-isobutylG, N(2)-benzylG, and N(2)-methyl(9-anthracenyl)G. Steady-state kinetics of single nucleotide incorporation by RT and T7(-) showed a decrease of 10(3) in k(cat)/K(m) for dCTP incorporation opposite N(2)-MeG and a further large decrease opposite N(2)-EtG. Misincorporation frequency was increased 10(2)-10(3)-fold by a Me group and another approximately 10(3)-fold by an Et group. dATP was preferentially incorporated opposite bulky N(2)-alkylG molecules. N(2)-MeG attenuated the pre-steady-state kinetic bursts with RT and T7(-), and N(2)-EtG eliminated the bursts. Large elemental effects with thio-dCTP(alphaS) were observed with N(2)-EtG (6- and 72-fold decreases) but were much less with N(2)-MeG, indicating that the N(2)-Et group may affect the rate of the chemistry step (phosphodiester bond formation). Similar values of K(d(dCTP)) and K(d(DNA)) and k(off) rates of DNA substrates from RT and T7(-) indicate that ground-state binding and dissociation rates are not considerably affected by the bulk. We conclude that even a Me group at the guanine N-2 atom can cause a profound interfering effect on the fidelity and efficiency; an Et or larger group causes preferential misincorporation and strong blockage of replicative polymerases, probably at and before the chemistry step, demonstrating the role of bulk in DNA lesions.

Alkylation↗
PubMed1991

Substrate specificity and kinetics of degradation of antisense oligonucleotides by a 3' exonuclease in plasma.

The pathways of degradation of oligodeoxynucleotides in plasma from several mammalian species, including human, were investigated. In all cases, hydrolysis occurred exclusively by a 3' to 5' exonucleolytic activity. Human, mouse, and rat plasma degraded oligonucleotides in this fashion at comparable rates, whereas rabbit plasma was severalfold more active. Single-stranded oligonucleotides were more susceptible to hydrolysis than double-stranded oligonucleotides. The rate of hydrolysis was sequence dependent: 3' pyrimidine nucleotides were cleaved more rapidly than 3' purines. The Km and Vmax values for an oligonucleotide 15-mer with the sequence TAGCACCATGGTTTC in human plasma were 50 microM and 4.5 microM/min, respectively. Substitution of the 3'-terminal phosphodiester internucleoside linkage with a phosphotriester rendered this substrate completely resistant to hydrolysis, showing that the enzyme is a pure 3' to 5' exonuclease and that there are no other nucleolytic activities in plasma. Modification at this position is required to inhibit rapid nuclease degradation of antisense compounds in vivo and in tissue culture systems requiring serum.

Animals↗
PubMed1997

Stability of stereoregular oligo(nucleoside phosphorothioate)s in human plasma: diastereoselectivity of plasma 3'-exonuclease.

The stability of stereoregular oligo(nucleoside phosphorothioate)s (PS-oligos) in human plasma has been studied. 3'-Exonuclease present in human plasma appeared to be RP specific, that is, it cleaves internucleotide phosphorothioate linkages of [RP]-configuration and not those of [SP]-configuration. Therefore, PS-oligos containing all phosphorothioate internucleotide linkages of [RP]-configuration [RP-PS-oligos]) are more effectively degraded by the enzyme than PS-oligos prepared via nonstereo-controlled methods (so-called random mixture of diastereomers [Mix-PS-oligos]), whereas oligo(nucleoside phosphorothioate)s of [S(P)]-configuration remain intact. The enzyme activity depends on the sequence of nucleobases. The presence of deoxycytidine units (three or more residues) at the 3'-end of PS-oligo substrate significantly inhibits the enzyme activity.

Drug Stability↗
PubMed1999

Real-time 5'-->3' exonuclease-based PCR assay for detection of the t(11;14)(q13;q32).

We describe the usefulness of a real-time polymerase chain reaction (PCR) assay for detection of the t(11;14)(q13;q32), most commonly present in mantle cell lymphoma (MCL). This assay is based on the 5'-->3' exonuclease activity of Taq polymerase, which cleaves an internal probe labeled with a reporter dye at its 5' end and a quencher dye at its 3' end during PCR. The real-time t(11;14) PCR assay was established using DNA from a case of MCL with the t(11;14), amplifiable using conventional PCR and primers specific for the major translocation cluster (MTC) region of the bcl-1 locus and the immunoglobulin heavy chain joining region gene (JH). The specificity was determined by analyzing DNA from 82 cases: 50 MCL, 27 other types of non-Hodgkin lymphoma (NHL), and 5 reactive lymphoid proliferations. The real-time t(11;14) PCR results were correlated with data obtained by a conventional PCR assay. By using the real-time assay, bcl-1 MTC/JH DNA fusion sequences were detected in 25 of 50 MCLs but not in other NHLs or reactive lymphoid proliferations. Concordance between real-time and conventional PCR methods for MCL was 96% and for all samples was 98%. The results demonstrate that this real-time PCR method to detect bcl-1 MTC/JH DNA fusion sequences is specific and reliable. In addition, the results are available immediately following amplification, without standard post-PCR manipulations.

Chromosomes, Human, Pair 11↗
PubMed1990

Physical analysis of spontaneous and mutagen-induced mutants of Escherichia coli K-12 expressing DNA exonuclease VIII activity.

We have mapped the extents of two deletion sbcA mutations which result in production of DNA exonuclease VIII (ExoVIII). One mutation, sbcA8, deletes about 140 kb of DNA which includes most of the Rac prophage and the trg gene. Western blot analysis shows that the protein produced is larger than wild type ExoVIII. The nucleotide sequence shows that a translational gene fusion has occurred. The N-terminal 294 codons of recE have been deleted and the remaining C-terminal codons have been fused to the N-terminal portion of another reading frame we call sfcA. Analysis of the protein sequence encoded by sfcA shows an 83% similarity with rat and mouse NADP-linked malic enzyme. We discuss the possibility that sfcA is identical to maeA which encodes NAD-linked malic enzyme from Escherichia coli. Restriction nuclease analysis of a second deletion, sbcA81, by Southern blot technique indicates that about 105 kb of DNA have been deleted and a transcriptional gene fusion has occurred between recE and the regulatory region of an E. coli chromosomal gene. We also examined eight other sbc mutations that result in ExoVIII production. Five have no effect on restriction nucleotide fragment sizes detected by complementarity to lambda rev as probe. These are presumed point mutations. Three seem to produce additional restriction nucleotide fragments complementary to lambda rev. The possible nature of these sbc mutations is discussed.

Amino Acid Sequence↗
PubMed1995

Suppression of recJ exonuclease mutants of Escherichia coli by alterations in DNA helicases II (uvrD) and IV (helD).

The recJ gene encodes a single-strand DNA-specific exonuclease involved in homologous recombination. We have isolated a pseudorevertant strain in which recJ mutant phenotypes were alleviated. Suppression of recJ was due to at least three mutations, two of which we have identified as alterations in DNA helicase genes. A recessive amber mutation, "uvrD517am," at codon 503 of the gene encoding helicase II was sufficient to suppress recJ partially. The uvrD517am mutation does not eliminate uvrD function because it affects UV survival only weakly; moreover, a uvrD insertion mutation could not replace uvrD517am as a suppressor. However, suppression may result from differential loss of uvrD function: mutation rate in a uvrD517am derivative was greatly elevated, equal to that in a uvrD insertion mutant. The second cosuppressor mutation is an allele of the helD gene, encoding DNA helicase IV, and could be replaced by insertion mutations in helD. The identity of the third cosuppressor "srjD" is not known. Strains carrying the three cosuppressor mutations exhibited hyperrecombinational phenotypes including elevated excision of repeated sequences. To explain recJ suppression, we propose that loss of antirecombinational helicase activity by the suppressor mutations stabilizes recombinational intermediates formed in the absence of recJ.

Adenosine Triphosphatases↗
PubMed1996

3'-->5' exonucleases of DNA polymerases epsilon and delta correct base analog induced DNA replication errors on opposite DNA strands in Saccharomyces cerevisiae.

The base analog 6-N-hydroxylaminopurine (HAP) induces bidirectional GC-->AT and AT-->GC transitions that are enhanced in DNA polymerase epsilon and delta 3'-->5' exonuclease-deficient yeast mutants, pol2-4 and pol3-01, respectively. We have constructed a set of isogenic strains to determine whether the DNA polymerases delta and epsilon contribute equally to proofreading of replication errors provoked by HAP during leading and lagging strand DNA synthesis. Site-specific GC-->AT and AT-->GC transitions in a Pol+, pol2-4 or pol3-01 genetic background were scored as reversions of ura3 missense alleles. At each site, reversion was increased in only one proofreading-deficient mutant, either pol2-4 or pol3-01, depending on the DNA strand in which HAP incorporation presumably occurred. Measurement of the HAP-induced reversion frequency of the ura3 alleles placed into chromosome III near to the defined active replication origin ARS306 in two orientations indicated that DNA polymerases epsilon and delta correct HAP-induced DNA replication errors on opposite DNA strands.

Adenine↗
PubMed2006

Endonuclease IV and exonuclease III are involved in the repair and mutagenesis of DNA lesions induced by UVB in Escherichia coli.

Exonuclease III (Exo III) and endonuclease IV (Endo IV) play a critical role in the base excision repair (BER) of Escherichia coli. Both are endowed with AP endonucleolytic activity, cleaving the 5' phosphodiester bond adjacent to spontaneous or induced abasic sites in DNA. Although mutants defective in Exo III (xthA) are usually hypersensitive to oxidative agents such as hydrogen peroxide, near-UV-light and X-rays, mutants defective in Endo IV (nfo) are not as sensitive as the xthA strain. To further investigate the roles of these AP endonucleases in DNA repair, we evaluated the sensitivity and mutagenesis of xthA and nfo strains after UVB and compared with UVC light. Our results revealed that xthA but not nfo strain was hypersensitive to UVB. The use of Fe(+2) ion chelator (dipyridyl), prior to irradiation, completely protected the xthA mutant against UVB lethal lesions, suggesting the generation of toxic oxidative lesions mediated by transition metal reactions. The nfo strain displayed increased UVB-induced mutagenesis, which was significantly suppressed by pre-treatment with dipyridyl. Although xthA strain did not display increased mutagenesis after UVC and UVB treatments, this phenotype was not related to xthA mutation, but rather to an unknown secondary mutation specifying an antimutator phenotype. After UVB irradiation, the base substitution spectra of nfo strain revealed a bias towards AT-->GC transitions and GC-->CG transversions, which were also suppressed by previous treatment with the iron chelator. Overall, on the basis of the differential sensitivities and mutational spectra displayed after UVC and UVB treatments, we propose a role for Endo IV and Exo III to counteract DNA damage induced by the oxidative counterpart of UVB in E.coli.

Chelating Agents↗
PubMed1988

The relative importance of Escherichia coli exonuclease III and endonuclease IV for the hydrolysis of 3'-phosphoglycolate ends in polydeoxynucleotides.

In vitro, in the presence of Mg++, the 3'-phosphoglycolatase activity of endonuclease IV is about 4-times smaller than that of exonuclease III for the same AP endonuclease activity. It thus seems that endonuclease IV has only a minor role in the repair of strand breaks limited by 3'-phosphoglycolate ends in Escherichia coli even after the amount of enzyme has been increased by induction with O2 -generating agents.

Bacterial Proteins↗
PubMed1998

Mutational analysis of the 3'-->5' proofreading exonuclease of Escherichia coli DNA polymerase III.

The epsilon subunit of Escherichia coli DNA polymerase III holoenzyme, the enzyme primarily responsible for the duplication of the bacterial chromosome, is a 3'-->5' exonuclease that functions as a proofreader for polymerase errors. In addition, it plays an important structural role within the pol III core. To gain further insight into how epsilon performs these joint structural and catalytic functions, we have investigated a set of 20 newly isolated dnaQ mutator mutants. The mutator effects ranged from strong (700-8000-fold enhancement) to moderate (6-20-fold enhancement), reflecting the range of proofreading deficiencies. Complementation assays revealed most mutators to be partially or fully dominant, suggesting that they carried an exonucleolytic defect but retained binding to the pol III core subunits. One allele, containing a stop codon 3 amino acids from the C-terminal end of the protein, was fully recessive. Sequence analysis of the mutants revealed mutations in the Exo I, Exo II and recently proposed Exo IIIepsilon motifs, as well as in the intervening regions. Together, the data support the functional significance of the proposed motifs, presumably in catalysis, and suggest that the C-terminus of straightepsilon may be specifically involved in binding to the alpha (polymerase) subunit.

Amino Acid Sequence↗
PubMed2006

Role of the tryptophan residue in the vicinity of the catalytic center of exonuclease III family AP endonucleases: AP site recognition mechanism.

The mechanisms by which AP endonucleases recognize AP sites have not yet been determined. Based on our previous study with Escherichia coli exonuclease III (ExoIII), the ExoIII family AP endonucleases probably recognize the DNA-pocket formed at an AP site. The indole ring of a conserved tryptophan residue in the vicinity of the catalytic site presumably intercalates into this pocket. To test this hypothesis, we constructed a series of mutants of ExoIII and human APE1. Trp-212 of ExoIII and Trp-280 of APE1 were critical to the AP endonuclease activity and binding to DNA containing an AP site. To confirm the ability of the tryptophan residue to intercalate with the AP site, we examined the interaction between an oligopeptide containing a tryptophan residue and an oligonucleotide containing AP sites, using spectrofluorimetry and surface plasmon resonance (SPR) technology. The tryptophan residue of the oligopeptide specifically intercalated into an AP site of DNA. The tryptophan residue in the vicinity of the catalytic site of the ExoIII family AP endonucleases plays a key role in the recognition of AP sites.

Amino Acid Sequence↗
PubMed2002

The effects of sequence length and oligonucleotide mismatches on 5' exonuclease assay efficiency.

Although increasingly used for DNA quantification, little is known of the dynamics of the 5' exonuclease assay, particularly in relation to amplicon length and mismatches at oligonucleotide binding sites. In this study we used seven assays targeting the c-myc proto-oncogene to examine the effects of sequence length, and report a marked reduction in efficiency with increasing fragment length. Three of the assays were further tested on 15 mammalian species to gauge the effect of sequence differences on performance. We show that the effects of probe and primer binding site mismatches are complex, with single point mutations often having little effect on assay performance, while multiple mismatches to the probe caused the greatest reduction in efficiency. The usefulness of the assays in predicting rates of 'allelic dropout' and successful polymerase chain reactions (PCRs) in microsatellite genotyping studies is supported, and we demonstrate that the use of a fragment more similar in size to typical microsatellites (190 bp) is no more informative than a shorter (81 bp) fragment. The assays designed for this study can be used directly for quantification of DNA from many mammalian species or, alternatively, information provided here can be used to design unique sequence-specific assays to maximise assay efficiency.

Animals↗
PubMed2004

Straightforward detection of SNPs in double-stranded DNA by using exonuclease III/nuclease S1/PNA system.

Single-nucleotide polymorphisms (SNPs) in double-stranded DNA (dsDNA) have been straightforwardly genotyped by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS). Peptide nucleic acid (PNA), a DNA analog, was used as a probe molecule. In its presence, genomic dsDNA was first treated with exonuclease III and then with nuclease S1. By these one-pot reactions, single-stranded DNA fragments including the SNP sites were formed in situ. These fragments were directly analyzed by MALDI-TOF MS, and the identity of the DNA base at the SNP site was determined in terms of mass number. By using two or more PNA probes simultaneously, multiplex analysis was also successful. Various genotypes of apolipoprotein E gene (epsilon2/epsilon2, epsilon3/epsilon3, epsilon4/epsilon4, epsilon2/epsilon3 and epsilon3/epsilon4) were identified from dsDNA obtained by PCR from corresponding patients.

Apolipoproteins E↗