遗传学实验技术

Type I DNA topoisomerases change the linking number of supercoiled DNA by carrying out orcheastrated cleavage and ligation reactions involving phosphodiester bonds. Strand cleavage is not the result of phosphodiester hydrolysis, but the result of transesterification of an acti ...

DNA topoisomerases I and II (topo I and topo II) are ubiquitous nuclear enzymes that regulate DNA topology by breaking and resealing one or both strands of a DNA duplex. Topoisomerase I makes a single-stranded break in a DNA duplex, mediates passage of the intact strand through the break, and then resea ...

The DNA unwinding effect of a drug, derived from either DNA intercalation or groove binding, represents a warning signal for its possible link to side effects or toxicity. DNA unwinding results in a lengthening of the double helix. This increase in length can be detected by physicochemical (such as ...

The interactions of small molecules with nucleic acids have provoked considerable interest in the field of antitumor drug design over the past three decades; however, critical information linking the physical-chemical properties associated with these complexes with their bio ...

One of the most powerful methods for investigating the molecular mechanism of drug action is the use of radioligand binding technique. The method allows investigators to pinpoint the direct target of the drug, and the nature of the interaction in terms of binding affinity, specificity, and coo ...

Fluoroquinolone antibacterials rapidly kill susceptible bacteria at clinically relevant drug concentrations; all evidence indicates that these drugs exert their lethality on Escherichia coli by inhibiting the normal function of the DNA gyrase (a bacterial topoisomerase ...

DNA Topoisomerases (topos) are ubiquitous enzymes that catalyze the breakage and rejoining of the DNA phosphodiester backbone, which together with an intervening strand passage event, allow this these enzymes to alter DNA topology (1,2). Intermediates in the strand passage reaction ...

Type I topoisomerases catalyze topological changes in duplex DNA by reversibly nicking one strand, whereas type II enzymes catalyze the transient breakage of both strands simultaneously. The type I enzymes alter the linking number of covalently closed circular DNA in steps of one, presu ...

Flow cytometry is a laser-based technique used for the quantitation of specific intracellular and extracellular properties of cells, bacteria, or other biological particles. It is unique in its ability to perform simultaneous multiparameter analysis and to sort single cell types fr ...

Topoisomerase II is a requisite enzyme that cleaves both strands of double-stranded DNA, allowing for passage of a second DNA duplex, resulting in the unwinding of chromosomal DNA. Topo II is covalently bound to the 5′-end of the cleaved DNA in a reversible reaction. However, treatment with numero ...

A variety of flow cytometric methods have been developed over the past 25 yr to study how treatment with chemotherapeutic agents affects cell-cycle progression. One of the most commonly used measurements relies on a singletime analysis of the DNA distribution of a cell population (1). This ana ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...