遗传学实验技术

The electrophoretic velocity of a duplex DNA ring is mainly determined by its overall shape. Consequently, DNA topoisomers of opposite supercoiling handedness can have identical gel velocity, and topoisomers highly supercoiled cannot be separated beyond some point. These proble ...

Agarose gel electrophoresis is by far the most widely used method for characterizing the topological state of DNA molecules. Although this technique has been used for more than 30 years, the physical mechanism underlying the resolution of topological states remains poorly understood. ...

DNA topoisomerases are enzymes that alter the topology of DNA. They have important functions in DNA replication, transcription, Holliday junction dissolution, chromosome condensation, and sister chromatid separation. Deficiencies in these enzymes are associated with dise ...

Eukaryotic type II topoisomerases (Topo II) are implicated in a wide range of cellular processes. Cells in which Topo II protein has been specifically depleted or mutated provide powerful systems for analysing the normal in vivo functions of Topo II proteins and for assessing their roles in var ...

Here, we describe methods to prepare a mammalian expression plasmid encoding EGFP fused to the amino-terminus of human DNA topoisomerase IIα (Topo IIα) for use in studying the dynamics of Topo IIα in living cells. In previous studies, this plasmid was transfected into LLC-Pk cells, a porcine epit ...

Posttranslational protein modification by the Small Ubiquitin-like MOdifiers (SUMO) is involved in many cellular functions including organization of nuclear structures and chromatin, transcriptional regulation, and nucleo-cytoplasmic transport. Both genetic and b ...

DNA topoisomerase II (Topo II), named Top2 in budding and fission yeast, is a conserved target of the SUMO modification pathway, with SUMO-conjugated forms of Topo II accumulating specifically during mitosis in both yeast and vertebrate cells (Bachant et al., Mol Cell 9, 1169–82, 2002; Azuma et al., J C ...

For analyzing chromosome structural defects that result from topoisomerase II (topo II) dysfunction, we have adapted classical cell cycle experiments, classical cytological techniques, and the use of a potent topo II inhibitor (ICRF-193). In this chapter, we describe in detail the prot ...

Topoisomerase II activity is crucial to maintain genome stability through the removal of catenanes in the DNA formed during DNA replication and scaffolding the mitotic chromosome. Perturbed Topo II activity causes defects in chromosome segregation due to persistent catenations ...

Inhibition of Topo II function using poisons and catalytic inhibitors triggers checkpoints that act to delay progression of G2 cells into mitosis. Topo II poisons induce Topo II-associated DNA double-strand breaks that activate ATM and the DNA damage G2 checkpoint. Topo II catalytic inhi ...

Exposure of cells to inhibitors of DNA topoisomerase I (topo I) or topoisomerase II (topo II) leads to DNA damage that often involves formation of DNA double-strand breaks (DSBs). DNA damage, particularly induction of DSBs, manifests by phosphorylation of histone H2AX on Ser-139 which is media ...

The interaction of DNA topology modifying enzymes with eukaryotic DNA replication origins can be detected with nucleotide precision exploiting the action of enzyme poisons specific for type I or type II DNA topoisomerases. Using the topoisomerase I poison camptothecin and the topoi ...

In vertebrate cells, DNA topoisomerase II (Topo II), named Top2 in yeast, localizes along chromosome axes early in mitosis and concentrates within centromeric chromatin during metaphase. The factors controlling these changes in enzyme distribution are largely unknown. Insight in ...

Here we describe an adapted ChIP-on-chip protocol for the analysis of DNA topoisomerase chromosomal binding in Saccharomyces cerevisiae cells. The ChIP-on-chip technique is based on the immunoprecipitation of crosslinked chromatin (ChIP, chromatin immunoprecipitation), ...

In eukaryotic cells, topoisomerase III forms an evolutionarily conserved complex with a RecQ family helicase and two OB-fold containing proteins, replication protein A (RPA) and RMI1. One role for this complex is to catalyze the completion of homologous recombination reactions in whi ...

The past few years have seen the application of single-molecule force spectroscopy techniques to the study of topoisomerases. Magnetic tweezers are particularly suited to the study of topoisomerases due to their unique ability to exert precise and straightforward control of the supe ...

Type II DNA topoisomerases are essential, ubiquitous enzymes responsible for performing vital functions in chromosome condensation and segregation and in regulating intracellular DNA supercoiling. Topoisomerase II (topo II) performs these DNA transactions by passing one ...

To assay the preferential binding of eukaryotic type IB topoisomerases to supercoiled DNA, two methods are described that make use of a catalytically inactive mutant form of the enzyme. In the gel shift assay, the preference for binding to supercoiled plasmid DNA is detected in the presence of li ...

Detecting localized RNA in bacteria is difficult due to the properties of RNA and the small size of the cell. Fluorescence in situ hybridization (FISH) has been an invaluable method for detecting and imaging RNA. In FISH, RNA is fixed in its native subcellular position through chemical cross-lin ...

Noncoding RNAs serve myriad functions in the cell, but their biophysical properties are not well understood. Calorimetry offers direct and label-free means for characterizing the ligand-binding and thermostability properties of these RNA. We apply two main types of calorimetry—i ...