By Osheroff N., Bjornsti M.A.
DNA Topoisomerase Protocols is an exceptional number of state of the art experimental protocols for investigating DNA constitution, topology, and DNA topoisomerase functionality. defined through specialist experimentalists who've perfected the suggestions, those time-proven equipment make the most of new ways to check alterations in DNA topology (linking quantity, knotting, catenation, and rest) and DNA constitution (bending), in addition to to evaluate chromosome constitution via FLP-mediated recombination and to research bacterial nucleoid constitution. state of the art protocols additionally aspect the expression and purification of bacterial, viral, and eukaryotic DNA topoisomerases from a number of resources, together with baculovirus, bacterial, yeast, and mammalian expression structures.
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Additional info for DNA Topoisomerase Protocols
Note that spot –14C has a first-dimensional mobility midway between that of spots –7 and –8. 5 turn change in the linking difference. 5). 2. Materials 1. A closed circular plasmid 2000–6000 bp in length containing a palindrome 50–80 bp in length. 2. Ethidium bromide dissolved in water to a concentration of 1 mg/mL and millipore-filtered. 3. 5, 40 mM EDTA, 400 mM NaCl, 4 mM DTT, 100 µg/mL BSA. 32 Courey Fig. 1. Analysis of cruciform formation in plasmid pAC103 by 2-D agarose gel electrophoresis.
If a DNA fragment is already curved in its linear form, its circularization will cause the molecule to adopt a highly preferred configuration (12,13). In the absence of intrinsic bending, circularization of a small DNA fragment leads to a random orientation of the DNA duplex around its axis. In this case, binding of a DNA-bending protein fixes the rotational orientation of the minicircle (13). 3. Analysis of Protein-Induced DNA Bending and Supercoiling When biophysical methods cannot be applied, the ability of a protein to mediate curvature in double-stranded DNA can be tested biochemically by the circular permutation assay (14,15) or alternatively, by examining the effect of the protein on the DNA ligase-dependent cyclization of very short DNA fragments.
The DNA topoisomerases play the central role in relaxing this supercoiling (1–4). The study of DNA topoisomerases and DNA topology relies essentially on a two-part system: the DNA topoisomerase and the molecule of DNA. The DNA topoisomerases function as enzymes because they efficiently form covalent complexes with DNA through an active site tyrosine (1–4). The enzymes then relax supercoiled DNA. The mechanism of DNA relaxation differs depending on the type of DNA topoisomerase examined. The DNA topoisomerase then religates the cleaved strand and dissociates from the DNA.