Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Escherichia coli exonuclease III and endonuclease III are two distinct DNA-repair enzymes that can cleave apurinic/apyrimidinic (AP) sites by different mechanisms. While the AP endonuclease activity of exonuclease III generates a 3'-hydroxyl group at AP sites, the AP lyase activity of endonuclease III produces a 3'-alpha,beta unsaturated aldehyde that prevents DNA-repair synthesis. Saccharomyces cerevisiae Apn1 is the major AP endonuclease/3'-diesterase that also produces a 3'-hydroxyl group at the AP site, but it is unrelated to either exonuclease III or endonuclease III. apn1 deletion mutants are unable to repair AP sites generated by the alkylating agent methyl methane sulphonate and display a spontaneous mutator phenotype. This work shows that either exonuclease III or endonuclease III can functionally replace yeast Apn1 in the repair of AP sites. Two conclusions can be derived from these findings. The first of these conclusions is that yeast cells can complete the repair of AP sites even though they are cleaved by AP lyase. This implies that AP lyase can contribute significantly to the repair of AP sites and that yeast cells have the ability to process the alpha,beta unsaturated aldehyde produced by endonuclease III. The second of these conclusions is that unrepaired AP sites are strictly the cause of the high spontaneous mutation rate in the apn1 deletion mutant.
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PMID:Normal processing of AP sites in Apn1-deficient Saccharomyces cerevisiae is restored by Escherichia coli genes expressing either exonuclease III or endonuclease III. 919 99

The deduced amino acid sequence of the open reading frame 1 (ORF1) of the L1Tc non-site-specific non-long terminal repeat retrotransposon of Trypanosoma cruzi exhibits a significant homology with the consensus sequence of the class II family of the endonuclease apurinic-apyrimidinic (AP) proteins. The analysis of the activity of the 40-kDa recombinant protein, named NL1Tc, obtained from the expression of the L1Tc ORF1 in an Escherichia coli "in vitro" expression system revealed that the sequence codes for a protein with endonuclease activity specific for apurinic-apyrimidinic (AP) sites. Data are also presented showing that in vivo expression of the NL1Tc protein conferred viability by complementation to E. coli exonuclease III deletion mutants (BW286 strain). We propose that the biological function of the AP endonuclease activity of the NL1Tc protein may be connected with the introduction into the DNA of free 3' ends that could be used as primers for the integration, along the T. cruzi genome, of the L1Tc element and that the nicking could be a general mechanism for the retrotransposition of non-site-specific non-long terminal repeat retrotransposons.
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PMID:The open reading frame 1 of the L1Tc retrotransposon of Trypanosoma cruzi codes for a protein with apurinic-apyrimidinic nuclease activity. 931 37

Apurinic-apyrimidinic (AP) sites are DNA lesions that lack template information and are produced either spontaneously or by a variety of DNA damaging agents. AP sites must therefore be repaired; otherwise they are mutagenic. All cells investigated to date possess the DNA repair enzyme AP endonuclease that can repair AP sites. There are two discrete families of AP endonucleases, Exo III and Endo IV, which are exemplified by Escherichia coli exonuclease III and endonuclease IV, respectively. These AP endonucleases have evolved not only to repair AP sites but also to correct distinct DNA lesions.
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PMID:The apurinic-apyrimidinic endonuclease IV family of DNA repair enzymes. 949 55

To discover the physiological role of the Bacillus subtilis ExoA protein, which is similar in amino acid sequence to Escherichia coli exonuclease III, an exoA::Cm disruption was constructed in the chromosomal DNA of B. subtilis. There was no clear difference in tolerance to hydrogen peroxide and alkylating agents between the disruptant and the wild type strain. An expression plasmid of the ExoA in E. coli was constructed by inserting the exoA gene into the expression vector pKP1500. The purified ExoA was used to clarify enzymatic characterizations using synthetic DNA oligomers as substrates. A DNA oligomer containing a 1', 2'-dideoxyribose residue as an AP site, a DNA-RNA chimera oligomer, and a 3' end 32P-labeled oligomer were synthesized. It has been shown that the ExoA has AP endonuclease, 3'-5' exonuclease, ribonuclease H, and 3'-phosphomonoesterase activities. Thus, it has been confirmed that ExoA is a multifunctional DNA-repair enzyme in B. subtilis that is very similar to E. coli exonuclease III except that ExoA has lower 3'-5' exonuclease activity than that of E. coli exonuclease III.
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PMID:Characterization of Bacillus subtilis ExoA protein: a multifunctional DNA-repair enzyme similar to the Escherichia coli exonuclease III. 1054 Jul 38

In order to elucidate the mechanism of AP site recognition by Echerichia coli exonuclease III (exoIII), site-directed mutagenesis of the Tyr109, the Trp212, and the Phe213 residues, which were conserved in the type II AP endonuclease from various organisms and located in the vicinity of the catalytic site, was performed. The exoIII-W212S mutant lacked any detectable AP endonuclease activity and binding ability to the duplex DNA containing an AP site, while the exoIII-Y109S and exoIII-F213W mutants retained a low level of activities (13% and 83%, respectively, compared with wild-type exoIII). This study suggests that the Trp212 is an important component for abasic site recognition by the E. coli exonuclease III.
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PMID:Abasic site recognition mechanism by the Escherichia coli exonuclease III. 1078 Apr 46

Abasic sites and non-conventional 3'-ends, e.g. 3'-oxidized fragments (including 3'-phosphate groups) and 3'-mismatched nucleotides, arise at significant frequency in the genome due to spontaneous decay, oxidation or replication errors. To avert the potentially mutagenic or cytotoxic effects of these chromosome modifications/intermediates, organisms are equipped with apurinic/apyrimidinic (AP) endonucleases and 3'-nucleases that initiate repair. Ape1, which shares homology with Escherichia coli exonuclease III (ExoIII), is the major abasic endonuclease in mammals and an important, yet selective, contributor to 3'-end processing. Mammals also possess a second protein (Ape2) with sequence homology to ExoIII, but this protein exhibits comparatively weak AP site-specific and 3'-nuclease activities. Prompted by homology modeling studies, we found that substitutions in the hydrophobic pocket of Ape1 (comprised of F266, W280 and L282) reduce abasic incision potency about fourfold to 450,000-fold, while introduction of an ExoIII-like pocket into Ape2 enhances its AP endonuclease function. We demonstrate that mutations at F266 and W280 of Ape1 increase 3' to 5' DNA exonuclease activity. These results, coupled with prior comparative sequence analysis, indicate that this active-site hydrophobic pocket influences the substrate specificity of a diverse set of sequence-related proteins possessing the conserved four-layered alpha/beta-fold. Lastly, we report that wild-type Ape1 excises 3'-mismatched nucleotides at a rate up to 374-fold higher than correctly base-paired nucleotides, depending greatly on the structure and sequence of the DNA substrate, suggesting a novel, selective role for the human protein in 3'-mismatch repair.
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PMID:Determinants in nuclease specificity of Ape1 and Ape2, human homologues of Escherichia coli exonuclease III. 1186 37

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.
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PMID:Role of the tryptophan residue in the vicinity of the catalytic center of exonuclease III family AP endonucleases: AP site recognition mechanism. 1654 May 94

Apurinic/apyrimidinic (AP) sites arise in DNA through the spontaneous loss of bases or through the release of damaged bases from DNA by DNA glycosylases. AP sites in DNA can be catalyzed by AP endonucleases such as exonuclease III and endonuclease IV, generating a 3'-hydroxyl group and a 5'-terminal sugar phosphate. Here, we have identified and characterized a novel endonuclease IV from a hyperthermophilic bacterium Thermus thermophilus designated as TthNfo. TthNfo efficiently removed AP site from double-stranded oligonucleotide substrate. No significant difference was observed in the rate of reaction of four bases opposite AP site with TthNfo. In addition, TthNfo possesses a 3'-5' exonuclease activity similar to that of Escherichia coli exonuclease III. Surprisingly, we found that TthNfo also catalyzes the excision of uracil from DNA. In comparison with other endonuclease IV proteins, the removal of uracil residue was unique to TthNfo. Based on these observations and the absence of exonuclease III in T. thermophilus, we suggest that versatile enzyme activities of TthNfo play an important role in counteracting DNA base damage in vivo.
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PMID:A versatile endonuclease IV from Thermus thermophilus has uracil-excising and 3'-5' exonuclease activity. 1678 61

Sweet potato (Ipomoea batatas) is an important subsistence and famine reserve crop grown in developing countries where Sweet potato chlorotic stunt virus (SPCSV; Closteroviridae), a single-stranded RNA (ssRNA) crinivirus, synergizes unrelated viruses in co-infected sweet potato plants. The most severe disease and yield losses are caused by co-infection with SPCSV and a potyvirus, Sweet potato feathery mottle virus (SPFMV; Potyviridae). Potyviruses synergize unrelated viruses by suppression of RNA silencing with the P1/HC-Pro polyprotein; however, the SPCSV-SPFMV synergism is unusual in that the potyvirus is the beneficiary. Our data show that transformation of an SPFMV-resistant sweet potato variety with the double-stranded RNA (dsRNA)-specific class 1 RNA endoribonuclease III (RNase3) of SPCSV broke down resistance to SPFMV, leading to high accumulation of SPFMV antigen and severe disease symptoms similar to the synergism in plants co-infected with SPCSV and SPFMV. RNase3-transgenic sweet potatoes also accumulated higher concentrations of 2 other unrelated viruses and developed more severe symptoms than non-transgenic plants. In leaves, RNase3 suppressed ssRNA-induced gene silencing (RNAi) in an endonuclease activity-dependent manner. It cleaved synthetic double-stranded small interfering RNAs (siRNAs) of 21, 22, and 24 bp in vitro to products of approximately 14 bp that are inactive in RNAi. It also affected total siRNA isolated from SPFMV-infected sweet potato plants, suggesting a viral mechanism for suppression of RNAi by cleavage of siRNA. Results implicate RNase3 in suppression of antiviral defense in sweet potato plants and reveal RNase3 as a protein that mediates viral synergism with several unrelated viruses, a function previously described only for P1/HC-Pro.
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PMID:Elimination of antiviral defense by viral RNase III. 1951 15

BspQI is a thermostable Type IIS restriction endonuclease (REase) with the recognition sequence 5'GCTCTTC N1/N4 3'. Here we report the cloning and expression of the bspQIR gene for the BspQI restriction enzyme in Escherichia coli. Alanine scanning of the BspQI charged residues identified a number of DNA nicking variants. After sampling combinations of different amino acid substitutions, an Nt.BspQI triple mutant (E172A/E248A/E255K) was constructed with predominantly top-strand DNA nicking activity. Furthermore, a triple mutant of BspQI (Nb.BspQI, N235A/K331A/R428A) was engineered to create a bottom-strand nicking enzyme. In addition, we demonstrated the application of Nt.BspQI in optical mapping of single DNA molecules. Nt or Nb.BspQI-nicked dsDNA can be further digested by E. coli exonuclease III to create ssDNA for downstream applications. BspQI contains two potential catalytic sites: a top-strand catalytic site (Ct) with a D-H-N-K motif found in the HNH endonuclease family and a bottom-strand catalytic site (Cb) with three scattered Glu residues. BlastP analysis of proteins in GenBank indicated a putative restriction enzyme with significant amino acid sequence identity to BspQI from the sequenced bacterial genome Croceibacter atlanticus HTCC2559. This restriction gene was amplified by PCR and cloned into a T7 expression vector. Restriction mapping and run-off DNA sequencing of digested products from the partially purified enzyme indicated that it is an EarI isoschizomer with 6-bp recognition, which we named CatHI (CTCTTC N1/N4).
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PMID:Engineering BspQI nicking enzymes and application of N.BspQI in DNA labeling and production of single-strand DNA. 1974 45


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