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At least 721 records · Page 40Linked to original sources
PubMed1999

The effect of the Saccharomyces cerevisiae endo-exonuclease NUD1 gene expression on the resistance of HeLa cells to DNA-damaging agents.

HeLa cells transiently transfected with a mammalian expression DNA vector expressing the Saccharomyces cerevisiae endo-exonuclease (EE) NUD1 gene have exhibited changes in cell survival frequencies after treatment with different DNA-damaging agents as compared to HeLa cells transfected with a control plasmid. The NUD1-transfected cells showed a dose-dependent increase in sensitivity to UV irradiation resulting in up to 58% decrease in cell survival. In response to gamma-irradiation NUD1 transfected cells featured an increased survival at doses equal to and greater than 2.0 Gy, reaching a maximum enhancement in survival frequency of 17%. At the same time, the NUD1-transfectants featured an increase in resistance to 0.25 microM-0.5 microM cis-platin (up to 58% increase in cell survival) and 1.0 mM EMS (11% increase). At higher concentrations of EMS NUD1 expression resulted in a decreased cell survival of the transfected cells (17% decrease for 2.5 mM EMS). No difference in cell survival frequencies between the NUD1-transfectants and the controls was observed after treatment with different concentrations of chlorambucil and mechlorethamine. These results suggest possible roles played by EEs in different DNA repair pathways--being stimulatory for the repair of certain types of DNA lesions, such as double strand breaks (DSBs), and interfering with the endogenous DNA repair systems for the repair of other types of lesions. Furthermore, these results also provide additional indirect evidence for the role of EEs in homologous recombination.

Alkylating Agents↗
PubMed1999

Transient expression of Saccharomyces cerevisiae endo-exonuclease NUD1 gene increases the frequency of extrachromosomal homologous recombination in mouse Ltk- fibroblasts.

Endo-exonucleases (EEs) are nucleolytic enzymes which have been shown to participate in the processes of DNA repair and recombination in eukaryotes. Recently, we have demonstrated that transient expression of Saccharomyces cerevisiae EE NUD1 gene in HeLa cells increased the resistance of the latter to ionizing radiation and cisplatin, suggesting the involvement of the NUD1 gene product in the recombination repair of double-strand breaks (DSB). Here, we report that transient expression of NUD1 results in up to 62% increase in the frequency of homologous recombination between two co-transfected linear plasmids in mouse Ltk- cells.

Animals↗
PubMed2000

Synthesis and study of a new adenine-acridine tandem, inhibitor of exonuclease III.

A new heterodimer adenine-chain-acridine containing a mixed amido-guanidinium linker chain was synthesized. To achieve the synthesis a new method of introduction of aminoalkyl chain at position 9 of adenine was designed. The heterodimer interacts specifically with the abasic sites in DNA and inhibits the major base excision repair enzyme in Escherichia coli, Exonuclease III.

Acridines↗
PubMed1998

The 3' to 5' exonuclease activity of Mre 11 facilitates repair of DNA double-strand breaks.

MRE11 and RAD50 are known to be required for nonhomologous joining of DNA ends in vivo. We have investigated the enzymatic activities of the purified proteins and found that Mre11 by itself has 3' to 5' exonuclease activity that is increased when Mre11 is in a complex with Rad50. Mre11 also exhibits endonuclease activity, as shown by the asymmetric opening of DNA hairpin loops. In conjunction with a DNA ligase, Mre11 promotes the joining of noncomplementary ends in vitro by utilizing short homologies near the ends of the DNA fragments. Sequence identities of 1-5 base pairs are present at all of these junctions, and their diversity is consistent with the products of nonhomologous end-joining observed in vivo.

Amino Acid Sequence↗
PubMed1998

A 5'-3' exonuclease activity involved in forming the 3' products of histone pre-mRNA processing in vitro.

Histone RNA 3' processing in vitro produces one or more 5' cleavage products corresponding to the mature histone mRNA 3' end, and a group of 3' cleavage products whose 5' ends are mostly located several nucleotides downstream of the mRNA 3' end. The formation of these 3' products is coupled to the formation of 5' products and dependent on the U7 snRNP and a heat-labile processing factor. These short 3' products therefore are a true and general feature of the processing reaction. Identical 3' products are also formed from a model RNA containing all spacer nucleotides downstream of the mature mRNA 3' end, but no sequences from the mature mRNA. Again, this reaction is dependent on both the U7 snRNP and a heat-labile factor. Unlike the processing with a full-length histone pre-mRNA, this reaction produces only 3' but no 5' fragments. In addition, product formation is inhibited by addition of cap structures at the model RNA 5' end, indicating that product formation occurs by 5'-3' exonucleolytic degradation. This degradation of a model 3' product by a 5'-3' exonuclease suggests a mechanism for the release of the U7 snRNP after processing by shortening the cut-off histone spacer sequences base paired to U7 RNA.

Animals↗
PubMed2005

Enzymatically amplified surface plasmon resonance imaging detection of DNA by exonuclease III digestion of DNA microarrays.

This paper describes a novel approach utilizing the enzyme exonuclease III in conjunction with 3'-terminated DNA microarrays for the amplified detection of single-stranded DNA (ssDNA) with surface plasmon resonance (SPR) imaging. When ExoIII and target DNA are simultaneously introduced to a 3'-terminated ssDNA microarray, hybridization adsorption of the target ssDNA leads to the direction-dependent ExoIII hydrolysis of probe ssDNA strands and the release of the intact target ssDNA back into the solution. Readsorption of the target ssDNA to another probe creates a repeated hydrolysis process that results over time in a significant negative change in SPR imaging signal. Experiments are presented that demonstrate the direction-dependent surface enzyme reaction of ExoIII with double-stranded DNA as well as this new enzymatically amplified SPR imaging process with a 16-mer target ssDNA detection limit of 10-100 pM. This is a 10(2)-10(3) improvement on previously reported measurements of SPR imaging detection of ssDNA based solely on hybridization adsorption without enzymatic amplification.

DNA↗
PubMed2000

In vitro replication of primer-templates containing benzo[a]pyrene adducts by exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment): effect of sequence context on lesion bypass.

The presence of benzo[a]pyrene diol epoxide (B[a]PDE) adducts in DNA is known to interfere with DNA replication. Kinetic studies of nucleotide insertion by exonuclease-deficient E. coli DNA polymerase I (Klenow fragment) across from either the (+)-trans- or the (+)-cis-B[a]P-N(2)-dG adduct in the 5'-CGT-3' sequence context indicated that the rate of nucleotide incorporation followed the order: dAMP > dGMP > dTMP > dCMP, which did not correlate with the mutational spectrum observed for these adducts in this sequence in E. coli (mostly G-->A transitions). Interestingly, a kinetic analysis of extension past the adduct showed that, unlike other sequences studied, the primer-template was extended best when dT was positioned at the 3'-terminus of the primer across from either a (+)-trans- or a (+)-cis-B[a]P-N(2)-dG adduct. In contrast, when the (+)-trans-B[a]P-N(2)-dG adduct was positioned in the 5'-TGC-3' sequence context, which gives predominantly G-->T mutations in E. coli, extension was detectable only when dA was positioned across from the adduct. These data provide the first in vitro evidence that may explain why G-->A transitions, rather than the G-->T transversions found in other sequences, are preferred in the 5'-CGT-3' sequence in vivo.

Base Sequence↗
PubMed2001

Miscoding potential of the N2-ethyl-2'-deoxyguanosine DNA adduct by the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I.

Acetaldehyde, a major metabolite of ethanol, reacts with dG residues in DNA, resulting in the formation of the N(2)-ethyl-2'-deoxyguanosine (N(2)-Et-dG) adduct. This adduct has been detected in lymphocyte DNA of alcohol abusers. To explore the miscoding property of the N(2)-Et-dG DNA adduct, phosphoramidite chemical synthesis was used to prepare site-specifically modified oligodeoxynucleotides containing a single N(2)-Et-dG. These N(2)-Et-dG-modified oligodeoxynucleotides were used as templates for primer extension reactions catalyzed by the 3' --> 5' exonuclease-free (exo(-)) Klenow fragment of Escherichia coli DNA polymerase I. The primer extension was retarded one base prior to the N(2)-Et-dG lesion and opposite the lesion; however, when the enzyme was incubated for a longer time or with increased amounts of this enzyme, full extension occurred. Quantitative analysis of the fully extended products showed the preferential incorporation of dGMP and dCMP opposite the N(2)-Et-dG lesion, accompanied by a small amounts of dAMP and dTMP incorporation and one- and two-base deletions. Steady-state kinetic studies were also performed to determine the frequency of nucleotide insertion opposite the N(2)-Et-dG lesion and chain extension from the 3' terminus from the dN.N(2)-Et-dG (N is C, A, G, or T) pairs. These results indicate that the N(2)-Et-dG DNA adduct may generate G --> C transversions in living cells. Such a mutational spectrum has not been detected with other methylated dG adducts, including 8-methyl-2'-deoxyguanosine, O(6)-methyl-2'-deoxyguanosine, and N(2)-methyl-2'-deoxyguanosine. In addition, N(2)-ethyl-2'-deoxyguanosine triphosphate (N(2)-Et-dGTP) was efficiently incorporated opposite a template dC during DNA synthesis catalyzed by the exo(-) Klenow fragment. The utilization of N(2)-Et-dGTP was also determined by steady-state kinetic studies. N(2)-Et-dG DNA adducts are also formed by the incorporation of N(2)-Et-dGTP into DNA and may cause mutations, leading to the development of alcohol- and acetaldehyde-induced human cancers.

DNA Adducts↗
PubMed2006

Intercalating polycyclic aromatic hydrocarbon-DNA adducts poison DNA religation by Vaccinia topoisomerase and act as roadblocks to digestion by exonuclease III.

Polycyclic aromatic hydrocarbon (PAH)-DNA adducts pervert the execution or fidelity of enzymatic DNA transactions and cause mutations and cancer. Here, we examine the effects of intercalating PAH-DNA adducts on the religation reaction of vaccinia DNA topoisomerase, a prototypal type IB topoisomerase (TopIB), and the 3' end-resection reaction of Escherichia coli exonuclease III (ExoIII), a DNA repair enzyme. Vaccinia TopIB forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a target site 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p / N(-1) in duplex DNA. The rate of the forward cleavage reaction is suppressed to varying degrees by benzo[a]pyrene (BP) or benzo[c]phenanthrene (BPh) adducts at purine bases within the 3'-G(+5)G(+4)G(+3)A(+2)A(+1)T(-1)A(-2) sequence of the nonscissile strand. We report that BP adducts at the +1 and -2 N6-deoxyadenosine (dA) positions flanking the scissile phosphodiester slow the rate of DNA religation to a greater degree than they do the cleavage rate. By increasing the cleavage equilibrium constant > or = 10-fold, the BPdA adducts, which are intercalated via the major groove, act as TopIB poisons. With respect to ExoIII, we find that (i) single BPdA adducts act as durable roadblocks to ExoIII digestion, which is halted at sites 1 and 2 nucleotides prior to the modified base; (ii) single BPhdA adducts, which also intercalate via the major groove, elicit a transient pause prior to the lesion, which is eventually resected; and (iii) BPh adducts at N2-deoxyguanosine, which intercalate via the minor groove, are durable impediments to ExoIII digestion. These results highlight the sensitivity of repair outcomes to the structure of the PAH ring system and whether intercalation occurs via the major or minor groove.

Base Sequence↗
PubMed1996

Kinetic mechanism of the 3'-->5' proofreading exonuclease of DNA polymerase III. Analysis by steady state and pre-steady state methods.

DNA polymerase III holoenzyme is the major replicative enzyme in Escherichia coli. An important component of the high-fidelity DNA synthesis that is characteristic of DNA polymerase III holoenzyme is the 3'-->5' proofreading exonuclease activity resident in the epsilon subunit. Steady state and pre-steady state conditions have been used to determine equilibrium and Michaelis constants for substrate binding and the rate constant for cleavage by purified epsilon subunit. The steady state kinetic constants are K(m) = 16 +/- 6 microM and kcat = 210 +/- 23 s-1 for degradation of single-stranded DNA by epsilon. These steady state values are in agreement with the rate constants determined for excision of the 3' nucleotide of a dT10 oligomer under pre-steady state conditions. Using a simple two-step model, E + Dn reversible E.Dn-->E + Dn-1, we find K = 12 microM and kf = 280 s-1 for the dT10 substrate. In these experiments, epsilon subunit acts in a distributive manner and product release is not the rate-limiting step. Activity of the epsilon subunit on paired DNA oligonucleotides with zero to three mismatches at the 3' terminus indicates that an additional step is required in the mechanism. In the scheme Dn reversible Dn* + E reversible E.Dn*-->E + Dn-1, the 3' terminus undergoes a conformational change or "melts" before the DNA is a substrate for epsilon subunit. With this additional step, the values for binding of activated substrate and cleavage are the same as those for single-stranded DNA. The kinetics for exonucleolytic degradation of single-stranded, paired, and mispaired oligonucleotides support the model that the rate-limiting step in exonucleolytic proofreading of DNA by epsilon subunit is the DNA-melting step.

Base Composition↗
PubMed2003

Construction of linear functional expression elements with DNA fragments created by site-specific DNA nickase, N.Bpu10 I, and exonuclease III.

A method to assemble linear expression elements for rapid gene expression is described. Primers containing target specific sequences and N.Bpu10 I nickase recognition sites were used to amplify promoter, open reading frame and terminator fragments. Amplified fragments were treated with N.Bpu10 I nickase and exonuclease III to generate overhangs for directional ligation. These fragments were ligated and further amplified with element-specific primers. The amplified DNA was transfected into mammalian cells for gene expression.

Animals↗
PubMed2004

The yeast Rat1 exonuclease promotes transcription termination by RNA polymerase II.

The carboxy-terminal domain (CTD) of the RNA polymerase II (RNApII) largest subunit consists of multiple heptapeptide repeats with the consensus sequence YSPTSPS. Different CTD phosphorylation patterns act as recognition sites for the binding of various messenger RNA processing factors, thereby coupling transcription and mRNA processing. Polyadenylation factors are co-transcriptionally recruited by phosphorylation of CTD serine 2 (ref. 2) and these factors are also required for transcription termination. RNApII transcribes past the poly(A) site, the RNA is cleaved by the polyadenylation machinery, and the RNA downstream of the cleavage site is degraded. Here we show that Rtt103 and the Rat1/Rai1 5' --> 3' exonuclease are localized at 3' ends of protein coding genes. In rat1-1 or rai1Delta cells, RNA 3' to polyadenylation sites is greatly stabilized and termination defects are seen at many genes. These findings support a model in which poly(A) site cleavage and subsequent degradation of the 3'-downstream RNA by Rat1 trigger transcription termination.

Chromatin Immunoprecipitation↗
PubMed2006

Mutations in the gene encoding the 3'-5' DNA exonuclease TREX1 cause Aicardi-Goutières syndrome at the AGS1 locus.

Aicardi-Goutières syndrome (AGS) presents as a severe neurological brain disease and is a genetic mimic of the sequelae of transplacentally acquired viral infection. Evidence exists for a perturbation of innate immunity as a primary pathogenic event in the disease phenotype. Here, we show that TREX1, encoding the major mammalian 3' --> 5' DNA exonuclease, is the AGS1 gene, and AGS-causing mutations result in abrogation of TREX1 enzyme activity. Similar loss of function in the Trex1(-/-) mouse leads to an inflammatory phenotype. Our findings suggest an unanticipated role for TREX1 in processing or clearing anomalous DNA structures, failure of which results in the triggering of an abnormal innate immune response.

Animals↗
PubMed2003

A gene encoding an RNase D exonuclease-like protein is required for post-transcriptional silencing in Arabidopsis.

Post-transcriptional gene silencing (PTGS) and the closely related phenomenon RNA interference (RNAi) result from the initial endonucleolytic cleavage of target mRNAs, which are then presumed to be completely hydrolyzed by exoribonucleases. To date, no plant genes required for PTGS are known to encode exoribonucleases. The Arabidopsis Werner Syndrome-like exonuclease (WEX) gene encodes an RNase D domain most similar to that in human Werner Syndrome protein (WRN), but lacks the RecQ helicase domain. It is also related to Caenorhabditis elegans mut-7, which is essential for RNAi, PTGS, and transposon activity. We isolated a loss-of-function mutant, wex-1, that showed greatly reduced expression of WEX mRNA and early flowering. Although wex-1 did not affect expression of a robust marker for transcriptional gene silencing (TGS), PTGS of a green-fluorescent-protein (GFP) reporter gene was blocked in wex-1 and restored by ectopic expression of WEX, indicating that WEX is required for PTGS but not TGS. Thus, members of the RNase D protein family are required for PTGS in both plants and animals. Interestingly, WEX has been shown to interact with an Arabidopsis RecQ helicase, suggesting that these proteins might comprise a functional equivalent of WRN.

Arabidopsis↗
PubMed1998

Cross-linked DNA duplexes--exonuclease stability and interaction with the nucleic transcription factor of the kappa light-chain enhancer (NF-kappaB).

Investigation of substrate properties of DNA duplexes with covalently connected strands has been performed. The kinetic measurements of the hydrolysis processes of these compounds by snake venom phosphodiesterase has been carried out. It has been shown that the exonuclease stability of cross-linked DNA duplexes is greatly increased as compared with analogs without linkages between strands. The interaction of cross-linked DNA duplexes with the p50 subunit of human nucleic transcription factor of the kappa light-chain enhancer (NF-kappaB) has been studied. The presence of linkages between strands did not prevent the binding of protein with DNA duplexes and, in specific cases, even promoted this process. The speculation of using cross-linked DNA duplexes as efficient decoys for DNA-binding proteins such as NF-kappaB from the point of view of sense technology are discussed.

Alkaline Phosphatase↗
PubMed2000

Polynucleotide phosphorylase functions both as a 3' right-arrow 5' exonuclease and a poly(A) polymerase in Escherichia coli.

In vitro, polynucleotide phosphorylase of Escherichia coli can both synthesize RNA by using nucleotide diphosphates as precursors and exonucleolytically degrade RNA in the presence of inorganic phosphate. However, because of the high in vivo concentration of inorganic phosphate in exponentially growing cells, it has been assumed that the enzyme works exclusively as an exonuclease. Here we demonstrate that, contrary to this prediction, polynucleotide phosphorylase not only synthesizes long, highly heteropolymeric tails in vivo, but also accounts for all of the observed residual polyadenylylation in poly(A) polymerase I deficient strains. In addition, the enzyme is responsible for adding the C and U residues that are found in poly(A) tails in exponentially growing cultures of wild type E. coli.

Base Sequence↗
PubMed1989

Exonuclease III and the catalase hydroperoxidase II in Escherichia coli are both regulated by the katF gene product.

The levels of both exonuclease III (exo III, product of xthA) and hydroperoxidase II (HP-II, product of katE) activity in Escherichia coli were influenced by a functional katF gene. The katF gene product is also necessary for synthesis of HP-II. Mutations in either katF or xthA, but not katE, result in sensitivity to H2O2 and near-UV (300-400 nm) radiation. Exo III, encoded by the xthA locus, recognizes and removes nucleoside 5'-monophosphates near apurinic and apyrimidinic sites in damaged DNA. Extracts of katF mutant strains had little detectable exo III activity. When a katF+ plasmid was introduced into the katF mutant, exo III activity exceeded wild-type levels. We propose that the katF gene is a trans-acting positive regulator of exo III and HP-II enzymes, both of which are involved in cellular recovery from oxidative damage.

Apurinic Acid↗
PubMed1993

Identification of a 3'-->5' exonuclease activity associated with human RNA polymerase II.

Human RNA polymerase II is shown to be associated with a 3'-->5' exonuclease activity that removes nucleoside 5'-monophosphates from the 3' end of the transcripts in isolated ternary complexes. This activity is stimulated by SII, a protein that acts as a transcription elongation factor in vitro. In addition, we show that another transcription factor, TFIIF, stimulates a competing pyrophosphorolysis reaction. These findings raise interesting questions about the roles of these activities in vivo, including the possibility that this RNA polymerase may proofread the nascent transcript.

Adenoviridae↗