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)

PI-SceI, a homing endonuclease of the LAGLIDADG family, consists of two domains involved in DNA cleavage and protein splicing, respectively. Both domains cooperate in binding the recognition sequence. Comparison of the structures of PI-SceI in the absence and presence of substrate reveals major conformational changes in both the protein and DNA. Notably, in the protein-splicing domain the loop comprising residues 53-70 and adopts a "closed" conformation, thus enabling it to interact with the DNA. We have studied the dynamics of DNA binding and subsequent loop movement by fluorescence techniques. Six amino acids in loop53-70 were individually replaced by cysteine and modified by fluorescein. The interaction of the modified PI-SceI variants with the substrate, unlabeled or labeled with tetramethylrhodamine, was analyzed in equilibrium and stopped-flow experiments. A kinetic scheme was established describing the interaction between PI-SceI and DNA. It is noteworthy that the apparent hinge-flap motion of loop53-70 is only observed in the presence of a divalent metal ion cofactor. Substitution of the major Mg2+-binding ligands in PI-SceI, Asp-218 and Asp-326, by Asn or "nicking" PI-SceI with trypsin at Arg-277, which interferes with formation of an active enzyme.substrate complex, both prevent the conformational change of loop53-70. Deletion of the loop inactivates the enzyme. We conclude that loop53-70 is an important structural element that couples DNA recognition by the splicing domain with DNA cleavage by the catalytic domain and as such "communicates" with the Mg2+ binding sites at the catalytic centers.
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PMID:DNA recognition by the homing endonuclease PI-SceI involves a divalent metal ion cofactor-induced conformational change. 1463 13

During V(D)J recombination, the RAG1 and RAG2 proteins form a complex and initiate the process of rearrangement by cleaving between the coding and signal segments and generating hairpins at the coding ends. Prior to ligation of the coding ends by DNA ligase IV/XRCC4, these hairpins are opened by the ARTEMIS/DNA-PKcs complex. ARTEMIS, a member of the metallo-beta-lactamase superfamily, shares several features with other family members that act on nucleic acids. ARTEMIS exhibits exonuclease and, in concert with DNA-PKcs, endonuclease activities. To characterize amino acids essential for its catalytic activities, we mutated nine evolutionary conserved histidine and aspartic acid residues within ARTEMIS. Biochemical analyses and a novel in vivo V(D)J recombination assay allowed the identification of eight mutants that were defective in both overhang endonucleolytic and hairpin-opening activities; the 5' to 3' exonuclease activity of ARTEMIS, however, was not impaired by these mutations. These results indicate that the hairpin-opening activity of ARTEMIS and/or its overhang endonucleolytic activity are necessary but its exonuclease activity is not sufficient for the process of V(D)J recombination.
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PMID:Functional and biochemical dissection of the structure-specific nuclease ARTEMIS. 1507 7

The benzetheno exocyclic adduct of the cytosine (C) base (pBQ-C) is a product of reaction between DNA and a stable metabolite of the human carcinogen benzene, p-benzoquinone (pBQ). We reported previously that the pBQ-C-containing duplex is a substrate for the human AP endonuclease (APE1), an enzyme that cleaves an apurinic/apyrimidinic (AP) site from double stranded DNA. In this work, using molecular dynamics simulation (MD), we provided a structural explanation for the recognition of the pBQ-C adduct by APE1. Molecular modeling of the DNA duplex containing pBQ-C revealed significant displacement of this adduct toward the major groove with pronounced kinking of the DNA at the lesion site, which could serve as a structural element recognized by the APE1 enzyme. Using 3 ns MD it was shown that the position of the pBQ-C adduct is stabilized by two hydrogen bonds formed between the adduct and the active site amino acids Asp 189 and Ala 175. The pBQ-C/APE1 complex, generated by MD, has a similar hydrogen bond network between target phosphodiester bond at the pBQ-C site and key amino acids at the active site, as in the crystallographically determined APE1 complexed with an AP site-containing DNA duplex. The position of the adduct at the enzyme active site, together with the hydrogen bond network, suggests a similar reaction mechanism for phosphodiester bond cleavage of oligonucleotide containing pBQ-C as reported for the AP site.
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PMID:Structural insights by molecular dynamics simulations into specificity of the major human AP endonuclease toward the benzene-derived DNA adduct, pBQ-C. 1515 53

The telomere-specific long interspersed nuclear element, TRAS1, encodes an endonuclease domain, TRAS1-EN, which specifically cleaves the telomeric repeat targets (TTAGG)n of insects and (TTAGGG)n of vertebrates. To elucidate the sequence-specific recognition properties of TRAS1-EN, we determined the crystal structure at 2.4-A resolution. TRAS1-EN has a four-layered alpha/beta sandwich structure; its topology is similar to apurinic/apyrimidinic endonucleases, but the beta-hairpin (beta10-beta11) at the edge of the DNA-binding surface makes an extra loop that distinguishes TRAS1-EN from cellular apurinic/apyrimidinic endonucleases. A protein-DNA complex model suggests that the beta10-beta11 hairpin fits into the minor groove, enabling interaction with the telomeric repeats. Mutational studies of TRAS1-EN also indicated that the Asp-130 and beta10-beta11 hairpin structure are involved in specific recognition of telomeric repeats.
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PMID:Crystal structure of the endonuclease domain encoded by the telomere-specific long interspersed nuclear element, TRAS1. 1524 45

DNA fragmentation/degradation is an important step for apoptosis. However, in unicellular organisms such as yeast, this process has rarely been investigated. In the current study, we revealed eight apoptotic nuclease candidates in Saccharyomyces cerevisiae, analogous to the Caenorhabditis elegans apoptotic nucleases. One of them is Tat-D. Sequence comparison indicates that Tat-D is conserved across kingdoms, implicating that it is evolutionarily and functionally indispensable. In order to better understand the biochemical and biological functions of Tat-D, we have overexpressed, purified, and characterized the S. cerevisiae Tat-D (scTat-D). Our biochemical assays revealed that scTat-D is an endo-/exonuclease. It incises the double-stranded DNA without obvious specificity via its endonuclease activity and excises the DNA from the 3'- to 5'-end by its exonuclease activity. The enzyme activities are metal-dependent with Mg(2+) as an optimal metal ion and an optimal pH around 5. We have also identified three amino acid residues, His(185), Asp(325), and Glu(327), important for its catalysis. In addition, our study demonstrated that knock-out of TAT-D in S. cerevisiae increases the TUNEL-positive cells and cell survival in response to hydrogen hyperoxide treatment, whereas overexpression of Tat-D facilitates cell death. These results suggest a role of Tat-D in yeast apoptosis.
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PMID:Search for apoptotic nucleases in yeast: role of Tat-D nuclease in apoptotic DNA degradation. 1565 35

Argonaute (Ago) proteins constitute a key component of the RNA-induced silencing complex (RISC). We report the crystal structure of Aquifex aeolicus Ago (Aa-Ago) together with binding and cleavage studies, which establish this eubacterial Ago as a bona fide guide DNA strand-mediated site-specific RNA endonuclease. We have generated a stereochemically robust model of the complex, where the guide DNA-mRNA duplex is positioned within a basic channel spanning the bilobal interface, such that the 5' phosphate of the guide strand can be anchored in a basic pocket, and the mRNA can be positioned for site-specific cleavage by RNase H-type divalent cation-coordinated catalytic Asp residues of the PIWI domain. Domain swap experiments involving chimeras of human Ago (hAgo1) and cleavage-competent hAgo2 reinforce the role of the PIWI domain in "slicer" activity. We propose a four-step Ago-mediated catalytic cleavage cycle model, which provides distinct perspectives into the mechanism of guide strand-mediated mRNA cleavage within the RISC.
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PMID:Crystal structure of A. aeolicus argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage. 1606 Nov 86

Apurinic/apyrimidinic (AP) sites are common mutagenic and cytotoxic DNA lesions that arise from the loss of normal bases. APE1, the major AP endonuclease of human cells, plays a central role in the repair of AP sites through both its endonuclease and phosphodiesterase activities. A common APE1 polymorphism, a T-->G transversion (Asp 148 Glu), was previously shown to be associated with risk of lung cancer, an association that was modified by cigarette smoking. To explore the association between APE1 genotype, smoking and bladder cancer risk, we examined data from an existing case-control study of bladder cancer patients (n = 239) and control individuals (n = 215) recruited from urology clinics at 2 hospitals in North Carolina. Genotype at the polymorphic site was determined using allele-specific primer extension reactions, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We found no overall association between APE1 genotype and bladder cancer risk. In stratified analyses, however, a positive association with risk was observed with an increasing number of Glu alleles among never smokers, but not among smokers (p-value for interaction = 0.005). We can speculate that small allelic differences that are apparent in never smokers are obscured by the large amount of DNA damage found in smokers. Given the lack of established biological mechanisms, and suboptimal numbers of subjects in some exposure categories, our findings should be interpreted cautiously.
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PMID:APE1 genotype and risk of bladder cancer: evidence for effect modification by smoking. 1642 70

The chemical mechanism of phosphodiester bond hydrolysis catalyzed by a junction-resolving enzyme has been investigated. Endonuclease I of phage T7 is a member of the nuclease superfamily of proteins that include many restriction enzymes, and the structure of the active site is very similar to that of BglI in particular. It contains three acidic amino acids that coordinate two divalent metal ions. Using mass spectrometry we have shown that endonuclease I catalyzes the breakage of the P-O3' bond, in common with restriction enzymes. We have found that the pH dependence of the hydrolysis reaction is log-linear, with a gradient of 0.9. Substitution of the scissile phosphate by an electrically neutral methylphosphonate significantly impairs the rate of bond cleavage. However, the introduction of chirally pure methylphosphonate groups shows that the effect of substitution of the proS oxygen atom is much greater than that for the proR. This is consistent with our current model of the structure of the DNA bound in the active site of endonuclease I, where the proS oxygen atom is coordinated directly to both metal ions as it is in BglI. The activity is also very sensitive to repositioning of the carboxylate groups of Asp 55 and Glu 65 in the active site, although some restoration of activity in endonuclease I E65D was observed in the presence of Mn2+ ions. A mechanism of hydrolysis consistent with all of these data is proposed.
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PMID:Mechanistic aspects of the DNA junction-resolving enzyme T7 endonuclease I. 1654 20

DNA damage, such as abasic sites and DNA strand breaks with 3'-phosphate and 3'-phosphoglycolate termini present cytotoxic and mutagenic threats to the cell. Class II AP endonucleases play a major role in the repair of abasic sites as well as of 3'-modified termini. Human cells contain two class II AP endonucleases, the Ape1 and Ape2 proteins. Ape1 possesses a strong AP-endonuclease activity and weak 3'-phosphodiesterase and 3'-5' exonuclease activities, and it is considered to be the major AP endonuclease in human cells. Much less is known about Ape2, but its importance is emphasized by the growth retardation and dyshematopoiesis accompanied by G2/M arrest phenotype of the APE2-null mice. Here, we describe the biochemical characteristics of human Ape2. We find that Ape2 exhibits strong 3'-5' exonuclease and 3'-phosphodiesterase activities and has only a very weak AP-endonuclease activity. Mutation of the active-site residue Asp 277 to Ala in Ape2 inactivates all these activities. We also demonstrate that Ape2 preferentially acts at mismatched deoxyribonucleotides at the recessed 3'-termini of a partial DNA duplex. Based on these results we suggest a novel role for human Ape2 as a 3'-5' exonuclease.
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PMID:Human Ape2 protein has a 3'-5' exonuclease activity that acts preferentially on mismatched base pairs. 1668 56

In this study, we demonstrated that the methyltransferase activity associated with Dam was essential for attenuation of Aeromonas hydrophila virulence. We mutated aspartic acid and tyrosine residues to alanine within the conserved DPPY catalytic motif of Dam and transformed the pBAD/damD/A, pBAD/damY/A, and pBAD/damAhSSU (with the native dam gene) recombinant plasmids into the Escherichia coli GM33 (dam-deficient) strain. Genomic DNA (gDNA) isolated from either of the E. coli GM33 strains harboring the pBAD vector with the mutated dam gene was resistant to DpnI digestion and sensitive to DpnII restriction endonuclease cutting. These findings were contrary to those with the gDNA of E. coli GM33 strain containing the pBAD/damAhSSU plasmid, indicating nonmethylation of E. coli gDNA with mutated Dam. Overproduction of mutated Dam in A. hydrophila resulted in bacterial motility, hemolytic and cytotoxic activities associated with the cytotoxic enterotoxin (Act), and protease activity similar to that of the wild-type (WT) bacterium, which harbored the pBAD vector and served as a control strain. On the contrary, overproduction of native Dam resulted in decreased bacterial motility, increased Act-associated biological effects, and increased protease activity. Lactone production, an indicator of quorum sensing, was increased when the native dam gene was overexpressed, with its levels returning to that of the control strain when the dam gene was mutated. These effects of Dam appeared to be mediated through a regulatory glucose-inhibited division A protein. Infection of mice with the mutated Dam-overproducing strains resulted in mortality rates similar to those for the control strain, with 100% of the animals dying within 2 to 3 days with two 50% lethal doses (LD50s) of the WT bacterium. Importantly, immunization of mice with a native-Dam-overproducing strain at the same LD50 did not result in any lethality and provided protection to animals after subsequent challenge with a lethal dose of the control strain.
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PMID:Mutations within the catalytic motif of DNA adenine methyltransferase (Dam) of Aeromonas hydrophila cause the virulence of the Dam-overproducing strain to revert to that of the wild-type phenotype. 1698 54


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