Supercoils in prokaryotic DNA restrained in vivo.

Abstract

Cells of Escherichia coli containing the plasmid F were 7-irradiated with various doses to introduce determined numbers of single-strand breaks in the F DNA. The cells were then incubated to permit repair of the breaks while DNA gyrase was inhibited with coumermycin to limit restoration of any relaxed supercoils. Repaired, covalently continuous F DNA was isolated and its superhelical density was measured by two different methods. Both indicated that a major part (50-60%) of the negative superhelical turn's were maintained in the repaired molecules, suggesting that the supercoils are restrained in vivo.In eukaryotic chromatin and chromosomes, the first order of DNA packaging seems to be attained via the nucleosome structure (for reviews, see refs. 1 and 2). The double-helical DNA in this particle is coiled through its interaction with the core histones into about 13/4 toroidal superhelical turns per core nucleosome (3, 4). Prokaryotic DNA has superhelical densities similar to those found in eukaryotes (5, 6), but thereis no clear evidence for restraint of the supercoils in structures analogous to the nucleosome. Histone-like proteins are found in prokaryotes (7,8), and their mode of interaction with DNA in prokaryotic chromosomes may have some similarity with nucleosomic histones (8, 9). Also, beaded structures having the appearance of nucleosomes have been seen in an electron microscope study of DNA from partially lysed bacteria (10).However, pure DNA can spontaneously form beaded structures in certain solvents (11), and electron microscopic evidence alone cannot be considered sufficient to establish the existence of the prokaryotic equivalent of a nucleosome.DNA gyrase seems to play a vital role in forming DNA supercoils in prokaryotic chromosomes. This enzyme can catalyze an ATP-driven reaction that has the topological effect of decreasing the linking number between the two continuous strands of a DNA double helix, thereby introducing negative superhelical turns into the DNA (12). When the DNA gyrase is inhibited by coumermycin (or oxolinic acid) in intact Escherichia coli cells, X DNA molecules, which are converted in vivo to the covalently circular form, attain only a small fraction of their normal superhelical density (13,14). This result indicates that the DNA gyrase activity is necessary for the formation (or the maintenance or both) of at least part of the DNA supercoils in prokaryotic cells. Thus, it seems possible that, in viwo, supercoils in prokaryotic DNA may not be stabilized by a nucleosome-like structure and that the Major reason for both the formation and maintenance (15) of DNA supercoils is the activity of DNA gyrase. Other alternatives include the possibility that DNA supercoils catalyzed by the DNA gyrase are subsequently restrained in a nucleosome-like structure by interactions involving molecules other than DNA gyrase (9). These two alternatives differ in that in the former case the double helix (17). After irradiation, the 32p-labeled cells kept at 0C were divided into several p...

Modeling the Lac repressor-operator assembly: The influence of DNA looping on Lac repressor conformation

“…The situation changes if the loop is incorporated in a plasmid that is subject to the supercoiling found in vivo (31). Negative supercoiling is known to enhance and stabilize loop formation of DNA with LacR (30).…”

DNA supercoiling is a fundamental regulatory principle in the control of bacterial gene expression

“…The writhing of the underwound duplex is described as negative supercoiling (Higgins and Vologodskii 2015). In a bacterium such as Escherichia coli, about half of the DNA supercoils are constrained by interaction with proteins (Bliska and Cozzarelli 1987;Pettijohn and Pfenninger 1980), but the energy in the unconstrained portion is available to drive DNA transactions (Booker et al 2010). The constrained and unconstrained regions of the chromosome are unlikely to be fixed, displaying a dynamism that reflects changes in growth rate, growth phase and changes in the nature and sizes of the populations of DNA binding proteins that decorate the DNA.…”

Growth Rate Toxicity Phenotypes and Homeostatic Supercoil Control Differentiate Escherichia coli from Salmonella enterica Serovar Typhimurium

“…Unconstrained supercoiling (S U ) causes torsion that stabilizes inter-and intramolecular triplex structures, left-handed Z-DNA, and cruciforms (35). In E. coli, S U and S C are distributed roughly 50:50 (5,43,71). To compare S U in E. coli and Salmonella, the Z-DNA-forming plasmid pRW478 was introduced in both organisms.…”