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 topoisomerase (topo) III enzymes are found in organisms ranging from bacteria to humans, yet the precise cellular function of these enzymes remains to be determined. We previously found that Drosophila topo IIIbeta can relax plasmid DNA only if the DNA is first hypernegatively supercoiled. To investigate the possibility that topo IIIbeta requires a single-stranded region for its relaxation activity, we formed R-loops and D-loops in plasmids. In addition to containing a single-stranded region, these R-loops and D-loops have the advantage of being covalently closed and supercoiled, thus allowing us to assay for supercoil relaxation. We found that topo IIIbeta preferentially cleaves, rather than relaxes, these substrates. The cleavage of the R-loops and D-loops, which is primarily in the form of nicking, occurs to a greater extent at a temperature that is lower than the optimal temperature for relaxation of hypernegatively supercoiled plasmid. In addition, the cleavage can be readily reversed by high salt or high temperature, and the products fail to enter the gel in the absence of proteinase K treatment and are not observed with an active-site Y332F mutant of topo IIIbeta, indicating that the cleavage is mediated by a topoisomerase. We mapped the cleavage to the unpaired strand within the loop region and found that the cleavage occurs along the length of the unpaired strand. These studies suggest that the topo III enzyme behaves as a structure-specific endonuclease in vivo, providing a reversible DNA cleavage activity that is specific for unpaired regions in the DNA.
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PMID:Preferential cleavage of plasmid-based R-loops and D-loops by Drosophila topoisomerase IIIbeta. 1204 41

Bean golden mosaic virus (BGMV) contains predominantly circular single-stranded DNA, but DNA isolation by treating virus with proteinase K results in conversion of the DNA to a linear form. The conversion is inhibited by as little as 0.025% sodium dodecyl sulfate but not by phenylmethylsulfonyl fluoride and does not occur when purified DNA is incubated with proteinase K. Protein is not involved in the covalent structure of BGMV DNA; the conversion appears to be caused by an endonuclease that copurifies with the virus and is resistant to proteinase K. BGMV contains a major capsid protein with an estimated molecular weight of 27,400. Two minor proteins are found, even in the most highly purified virus preparations; their origin and role in virus structure or function are unknown. BGMV particles contain 19% DNA; therefore each geminate particle must contain a single DNA molecule, of which over 90% are circular. The estimated molecular weight of the virus is 3.8 x 10(6).
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PMID:The composition of bean golden mosaic virus and its single-stranded DNA genome. 1863 17

Abelmoschus esculentus (okra) is one of the polysaccharide rich crop plants. The polysaccharides interfere with nucleic acids and protein isolation thereby affecting the downstream molecular analysis. So, to understand the molecular systematics of okra, high quality DNA, RNA and proteins are essential. In this study we present a method for extracting genomic DNA, RNA and proteins from polysaccharide rich okra tissues. The conventional extraction procedures were integrated with purification treatments with pectinase, RNase and proteinase K, which improved the quality and quantity of DNA as well. Using SDS, additional washes with CIA and NaCl precipitation improved the RNA isolation both quantitatively and qualitatively. Finally, ammonium acetate mediated protein precipitation and re-solubilization increased the quality of total protein extracts from the okra leaves. All of the methods above not only eliminated the impurities but also improved the quality and quantity of nucleic acids and proteins. Further, we subjected these samples to versatile downstream molecular analyses such as restriction endonuclease digestion, RAPD, Southern, reverse transcription-PCR and Western analysis and were proved to be successful.
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PMID:Polysaccharide-free nucleic acids and proteins of Abelmoschus esculentus for versatile molecular studies. 2311 48

Chromosome conformation capture (3C) is a method for studying chromosomal organization that takes advantage of formaldehyde cross-linking to measure the spatial association of two pieces of chromatin. The 3C method begins with whole-cell formaldehyde fixation of chromatin. After cell lysis, solubilized chromatin is digested with a type II restriction endonuclease, and cross-linked DNA fragments are ligated together. Cross-links are reversed by degradation with proteinase K, and chimeric DNA molecules are purified by standard phenol:chloroform extraction. The resulting 3C library represents chromatin fragments that may be separated by large genomic distances or located on different chromosomes, but are close enough in three-dimensional space for cross-linking. Locus-specific oligonucleotide primers are used to detect interactions of interest in the 3C library using end-point polymerase chain reaction (PCR).
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PMID:Chromosome Conformation Capture (3C) in Budding Yeast. 2603 4

Through X-ray crystallographic fragment screening, 4-bromopyrazole was discovered to be a 'magic bullet' that is capable of binding at many of the ligand 'hot spots' found in HIV-1 reverse transcriptase (RT). The binding locations can be in pockets that are 'hidden' in the unliganded crystal form, allowing rapid identification of these sites for in silico screening. In addition to hot-spot identification, this ubiquitous yet specific binding provides an avenue for X-ray crystallographic phase determination, which can be a significant bottleneck in the determination of the structures of novel proteins. The anomalous signal from 4-bromopyrazole or 4-iodopyrazole was sufficient to determine the structures of three proteins (HIV-1 RT, influenza A endonuclease and proteinase K) by single-wavelength anomalous dispersion (SAD) from single crystals. Both compounds are inexpensive, readily available, safe and very soluble in DMSO or water, allowing efficient soaking into crystals.
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PMID:Rapid experimental SAD phasing and hot-spot identification with halogenated fragments. 2687 Mar 81

Increasing interest and application of recombinant adeno-associated viruses (rAAVs) in basic and clinical research have urged efforts to improve rAAV production quality and yield. Standard vector production workflows call for genome titration of purified vectors at the endpoint of production to assess yield. Unfortunately, quality control measures for preparations during mid-production steps and economical means to assess the fidelity of multiple batches of rAAV preparations are lacking. Here we describe a scalable and accurate method for the direct quantitative polymerase chain reaction (qPCR) titration of rAAV genomes in crude lysate. Lysate samples are pretreated with DNase I to remove vector and packaging plasmid DNAs, followed by proteinase K to release endonuclease-resistant packaged viral genomes and to proteolyze factors inherent to crude lysates that can impinge upon quantitative PCR efficiencies. We show that this method is precise, scalable, and applicable for vector genome titrations of both single-stranded and self-complementary AAV genomes irrespective of serotype differences-a major limitation for standard lysate transduction methods that indirectly screen for vector packaging efficiency. Our described method therefore represents a significant improvement to rAAV vector production in terms of alleviating time and cost burdens, in-process quality control assessment, batch/lot monitoring in large-scale preparations, and good manufacturing practices.
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PMID:A Scalable and Accurate Method for Quantifying Vector Genomes of Recombinant Adeno-Associated Viruses in Crude Lysate. 2848 44


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