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)

The synthesis of influenza virus mRNA requires primers generated by cleavage of host cell transcripts 10-13 nucleotides from the 5' end by a virally encoded endonuclease. This novel enzyme is an attractive target for the development of antiviral agents. An assay for the influenza virus endonuclease has been developed that monitors the substrate cleavage reaction only at the correct position in the sequence, thereby discriminating against nonspecific RNA cleavage products. The influenza endonuclease assay is sensitive enough to detect 200 amol of product. The assay employs a DNA polymerase-catalyzed extension of the endonuclease cleavage product using radiolabeled dGTP and a DNA template containing a 3' region complementary to the product joined to a 5' region consisting of 10 dC residues. The influenza endonuclease assay does not involve gel electrophoretic separation and is amenable to high volume screening of potential inhibitors. The assay may also be employed to determine the site of influenza endonucleolytic cleavage in the substrate.
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PMID:Assay for influenza virus endonuclease using DNA polymerase extension of a specific cleavage product. 859 78

We have studied mutagenic specificity of an abasic site by the yeast-transformation procedure using an oligonucleotide containing a single furan-type abasic site. The recipient yeast used was deficient in the major AP endonuclease (apn1). Sequence analysis of the transformants suggested that dATP was incorporated most frequently opposite the abasic site, while dGTP seemed to be incorporated opposite the abasic site in the recipient proficient in apn1. To explore the mechanism of this oligonucleotide transformation, we have also analyzed the transformation with phosphorothioate oligonucleotides with mismatched 3'-end. The results are discussed.
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PMID:Yeast oligonucleotide transformation: its mechanism and application to analysis of mutations induced by defined DNA lesions. 958 13

The adeno-associated virus (AAV) nonstructural proteins Rep68 and Rep78 are site-specific DNA binding proteins, ATP-dependent site-specific endonucleases, helicases, and ATPases. These biochemical activities are required for viral DNA replication and control of viral gene expression. In this study, we characterized the biochemical properties of the helicase and ATPase activities of homogeneously pure Rep68. The enzyme exists as a monomer in solution at the concentrations used in this study (<380 nM), as judged by its mobility in sucrose density gradients. Using a primed single-stranded (ss) circular M13 substrate, the helicase activity had an optimum pH of 7 to 7.5, an optimum temperature of 45 degreesC, and an optimal divalent-cation concentration of 5 mM MgCl2. Several nucleoside triphosphates could serve as cofactors for Rep68 helicase activity, and the order of preference was ATP = GTP > CTP = dATP > UTP > dGTP. The Km values for ATP in both the DNA helicase reaction and the site-specific trs endonuclease reaction were essentially the same, approximately 180 microM. Both reactions were sigmoidal with respect to ATP concentration, suggesting that a dimer or higher-order multimer of Rep68 is necessary for both DNA helicase activity and terminal resolution site (trs) nicking activity. Furthermore, when the enzyme itself was titrated in the trs endonuclease and ATPase reactions, both activities were second order with respect to enzyme concentration. This suggests that a dimer of Rep68 is the active form for both the ATPase and nicking activities. In contrast, DNA helicase activity was linear with respect to enzyme concentration. When bound to ssDNA, the enzyme unwound the DNA in the 3'-to-5' direction. DNA unwinding occurred at a rate of approximately 345 bp per min per monomeric enzyme molecule. The ATP turnover rate was approximately 30 to 50 ATP molecules per min per enzyme molecule. Surprisingly, the presence of DNA was not required for ATPase activity. We estimated that Rep translocates processively for more than 1,300 bases before dissociating from its substrate in the absence of any accessory proteins. DNA helicase activity was not significantly stimulated by substrates that have the structure of a replication fork and contain either a 5' or 3' tail. Rep68 binds only to ssDNA, as judged by inhibition of the DNA helicase reaction with ss or double-stranded (ds) DNA. Consistent with this observation, no helicase activity was detected on blunt-ended ds oligonucleotide substrates unless they also contained an ss 3' tail. However, if a blunt-ended ds oligonucleotide contained the 22-bp Rep binding element sequence, Rep68 was capable of unwinding the substrate. This means that Rep68 can function both as a conventional helicase for strand displacement synthesis and as a terminal-repeat-unwinding protein which catalyzes the conversion of a duplex end to a hairpin primer. Thus, the properties of the Rep DNA helicase activity suggest that Rep is involved in all three of the key steps in AAV DNA replication: terminal resolution, reinitiation, and strand displacement.
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PMID:Biochemical characterization of adeno-associated virus rep68 DNA helicase and ATPase activities. 988 64

In mammalian cells, more than one genome in a single cell has to be maintained throughout the entire life of the cell, namely, one in the nucleus and the other in the mitochondria. The genomes and their precursor nucleotides are highly exposed to reactive oxygen species, which are inevitably generated as a result of the respiratory function in mitochondria. To counteract such oxidative damage in nucleic acids, cells are equipped with several defense mechanisms. Modified nucleotides in the nucleotide pools are hydrolyzed, thus avoiding their incorporation into DNA or RNA. Damaged bases in DNA with relatively small chemical alterations are mainly repaired by the base excision repair (BER) system, which is initiated by the excision of damaged bases by specific DNA glycosylases. MTH1 protein hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-dGTP, 8-oxo-dATP, and 2-hydroxy (OH)-dATP to the monophosphates, and MTH1 are located in the cytoplasm, mitochondria, and nucleus. We observed an increased susceptibility to spontaneous carcinogenesis in Mth1-deficient mice and an alteration of MTH1 expression along with the accumulation of 8-oxo-dG in patients with various neurodegenerative diseases. Enzymes for the BER pathway, namely, 8-oxoG DNA glycosylase (OGG1), 2-OH-A/adenine DNA glycosylase (MUTYH), and AP endonuclease (APEX2) are also located both in the mitochondria and in the nuclei, and the expression of mitochondrial OGG1 is altered in patients with various neurodegenerative diseases. We also observed increased susceptibilities to spontaneous carcinogenesis in OGG1 and MUTYH-deficient mice. The increased occurrence of lung tumor in OGG1-deficient mice was completely abolished by the concomitant disruption of the Mth1 gene.
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PMID:Biological significance of the defense mechanisms against oxidative damage in nucleic acids caused by reactive oxygen species: from mitochondria to nuclei. 1512 88

Sensitive and selective detection of point mutation is essential to molecular biology research and early clinical diagnosis. Here, we demonstrate a single quantum dot (QD)-based biosensor for DNA point mutation assay. In this assay, a mutant target (G/C) remains unchanged after the endonuclease treatment, and the polymerase chain reaction (PCR) may be initiated with the assistance of primers and polymerase, generating a large number of mutant targets. The amplified mutant targets can be captured by biotinylated probes during the process of denaturation and annealing, and Cy5-dGTP may be assembled into the biotinylated probe with the catalysis of polymerase, leading to the formation of Cy5-labeled biotinylated probes. The Cy5-labeled biotinylated probes can be further assembled onto the QD surface to obtain a Cy5-DNA-QD complex, resulting in the generation of fluorescence resonance energy transfer (FRET) between the QD donor and the Cy5 receptor. The mutant targets can be quantitatively evaluated by the measurement of Cy5 counts by total internal reflection fluorescence (TIRF) microscopy. While in the presence of wild-type targets (T/A), no Cy5-dGTP can be assembled into the biotinylated probe due to the presence of a mismatch and consequently no FRET is observed. This single QD-based biosensor exhibits high sensitivity with a detection limit of 5.3 aM (or 32 copies) and can even discriminate as low as 0.01% variant frequency from the mixture of mutant targets and wild-type ones. Importantly, this biosensor can be used for genomic analysis in human lung cancer cells, and may be further applied for an early clinical diagnosis and personalized medicine.
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PMID:A single quantum dot-based biosensor for DNA point mutation assay. 2622 72

DNA methyltransferase (MTase) and polynucleotide kinase (PNK) are both DNA-dependent enzymes that play important roles in DNA methylation and DNA repair processes, respectively. Dysregulation of their activities is associated with various human diseases. Herein, we present a specific and sensitive biosensing strategy, named terminal deoxynucleotidyl transferase (TdT)-activated nicking enzyme amplification reaction (TdT-NEAR), for their activity detection. As for MTase detection, an enclosed dumbbell-shaped oligonucleotide substrate, whose symmetric stem containing a recognition site of Dam MTase and an incomplete recognition sequence of nicking endonuclease Nt.BbvCI, was used. Typically, the substrate is methylated by Dam MTase and subsequently cleaved by Dpn I. In the presence of TdT and dGTP, poly(guanine, G) sequences are extended from the released 3'-OH ends, achieving the conversion of the incomplete Nt.BbvCI recognition sequence to an intact one. The extension products can then be used to trigger Nt.BbvCI-catalyzed cyclic cleavage of fluorophore/quencher-labelled oligonucleotide probe, giving a significantly enhanced fluorescence output. Such a sensing system can achieve sensitive and specific detection of Dam MTase with a detection limit of 0.002 U/mL. The unique working mechanism endows the sensing system with improved anti-interference capability and thus increased application potential in complex biological samples. Moreover, it was also demonstrated to work well for Dam MTase inhibitor screening and inhibitory activity evaluation, thus holding great potential in disease diagnosis and drug discovery. Using a simpler 3'-phosphorylated linear substrate and the same fluorescent probe, the TdT-NEAR strategy can be easily extended to the activity analysis of PNK, thus revealing wide application potential in bioanalysis.
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PMID:Terminal deoxynucleotidyl transferase-activated nicking enzyme amplification reaction for specific and sensitive detection of DNA methyltransferase and polynucleotide kinase. 3153 51


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