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Sedimentation Analysis of Bacterial Nucleoid Structure

The physiology of bacterial DNA topoisomerases can be studied by examining how perturbation of intracellular enzyme activities affects the structure of extracted nucleoids. Since the few DNA nicks that occur when nucleoids are isolated (1,2) are localized by the presence of 50–100 barr ...

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Methods for Analyzing DNA Bending

DNA bending is observed in all DNA transactions, including replication, transcription, recombination, repair, and packaging. DNA bending can be sequence-directed, as in kinetoplast minicircle DNA and many synthetic sequences, or protein-induced, as in the nucleosome and in prote ...

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Coating DNA with RecA Protein to Distinguish DNA Path by Electron Microscopy

To understand, at the molecular level, the mechanism of enzymes that act on DNA, it is highly informative to know the topology of their substrates and products. To describe fully the topology of a DNA knot or catenane, it is necessary to know the overpassing and underpassing segments when two DNA helices ...

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Formation of Extrachromosomal DNA Rings in Saccharomyces cerevisiae Using Site-Specific Recombination

A hindrance to the study of structure and function of DNA elements is that sites of interest always lie within the context of other DNA sequences. This is particularly limiting when attempting to examine elements embedded within chromosomes inside intact cells. Analysis of the Saccharomyc ...

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Overexpression and Purification of Bacterial DNA Topoisomerase I

In order to carry out studies on the structure and mechanism of enzymes, substantial quantities of purified proteins are often needed for many of the commonly used biophysical methods. This is especially true for three-dimensional structure determination using X-ray crystallograp ...

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Overexpression and Purification of Bacterial DNA Gyrase

DNA gyrase is the bacterial type II topoisomerase that can introduce negative supercoils into DNA using the free energy of ATP hydrolysis (1,2). The enzyme from Escherichia coli consists of two proteins, A and B (termed GyrA and GyrB), of molecular masses 97 and 90 kDa, respectively; the active enzyme is ...

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Overexpression and Purification of Escherichia coliDNA Topoisomerase III

The development of recombinant DNA techniques and protein expression systems has been critical to the understanding of the structure and catalytic mechanism of topoisomerases. The ability to overexpress and purify large quantities of these molecules has led to the elucidation of the ...

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Purification of the Bacteriophage T4 Type II DNA Topoisomerase

Bacteriophage T4 encodes a type II topoisomerase with properties more similar to those of the eukaryotic class of enzymes than to those of the bacterial DNA gyrase (1,2). Indeed, the discovery of the T4 topoisomerase provided the first example of an ATP-dependent relaxing enzyme (3,4), and an unde ...

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Overexpression and Purification of Bacterial Topoisomerase IV

Escherichia coli topoisomerase IV (topo IV) was discovered by Kato et al. (1), who showed that the predicted open reading frames from the parC and parE genes encoded proteins with a high degree of amino acid similarity to gyrA and gyrB, respectively. A new superhelical DNA relaxation activity could be ...

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Introduction to DNA Topoisomerases

The helical structure of duplex DNA allows for the faithful duplication and transmission of genetic information from one generation to the next, at the same time maintaining the integrity of the polynucleotide chains. The complementary nature of the two antiparallel DNA strands enables ...

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Overexpression and Purification of DNA Topoisomerase I from Yeast

The mechanism of action of DNA topoisomerase I in catalyzing the relaxation of supercoiled DNA and how this reaction is perturbed by the antitumor drug camptothecin have been the subject of intense investigation (reviewedin (1-3). Much of this effort has focused on structure/function stu ...

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Overexpression and Purification of Saccharomyces cerevisiae DNA Topoisomerase II from Yeast

Mechanistic and structural studies require large quantities of highly purified enzyme. Unfortunately, traditional expression strategies using Escherichia coli are often not successful for eukaryotic proteins, especially large ones. Despite numerous attempts, overe ...

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Purification of DNA Topoisomerase II from Drosophila melanogaster

To characterize properly protein function and enzymatic activity, it is highly desirable to perform experiments with purified protein preparations. This is especially true in the case of topoisomerase II, because many of the enzymatic assays critical to the topoisomerase field (su ...

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Purification of DNA Topoisomerase I from Human Placenta

DNA topoisomerase I (topo I) has been firmly established as the molecular target of the camptothecin group of anticancer drugs. These drugs include camptothecin, topotecan, 9-amino-camptothecin, and irinotecan (1). Many of them are now in clinical trials and are showing activity against a ...

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Purification of Baculovirus-Expressed Human DNA Topoisomerase I

DNA topoisomerase I (topo I) can be isolated from cultured human cells in quantities that are more than sufficient for investigations into the ability of topo I to relax supercoiled DNA (250 μg/109 cells) (1,2). However, the production of human topo I (htopo I) in this manner becomes both costly and labor ...

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Metabolic Labeling, Immunoprecipitation, and Two-Dimensional Tryptic Phosphopeptide Mapping of Human Topoisomerase II

Protein phosphorylation is almost certainly the most important posttranslational mechanism of enzyme regulation in eukaryotic cells (reviewed in 1). The equilibrium between phosphorylation by protein kinases and dephosphorylation by protein phosphatases modulates t ...

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Visualization of DNA Topoisomerases by Electron Microscopy

Eukaryotic DNA Topoisomerase II (Topo II) is an essential enzyme that catalyzes the relaxation of supercoiled DNA and the segregation of newly replicated chromosomes (1-3). The enzyme is highly conserved through evolution, and appears to result from the fusion of the A- and B-subunits of bact ...

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Immunoblot Analysis and Band Depletion Assays

Western blotting has been widely utilized to detect various polypeptides or polypeptide epitopes (e.g., posttranslational modifications) within cells (reviewed in 1-4). If the signals in samples being analyzed are compared to a suitable standard curve and appropriate internal st ...

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Appendix: Compendium of DNA Topoisomerase Sequences

DNA topoisomerases can be grouped in to three families based on biochemical properties and amino acid sequence. Following are multiple protein sequence alignments of the members of each of these families: the eukaryotic DNA topoisomerase I type, the DNA topoisomerase II type, and the DNA top ...

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Two-Dimensional Agarose-Gel Electrophoresis of DNA Topoisomers

Gel electrophoresis in one dimension is often insufficient to distinguish various molecular species of DNA, since different conformers, and sometimes DNA molecules of totally different structures may have the same electrophoretic mobility. These DNA species can be resolved by two ...

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