Early melting of supercoiled DNA topoisomers observed by TGGE.

P.J. Safarik University, Faculty of Sciences, Department of Biochemistry, Moyzesova 11, 041 54 Koice, Slovakia and Institute of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, 043 53 Koice, Slovakia.
Nucleic Acids Research (Impact Factor: 9.11). 06/2000; 28(11):E51.
Source: PubMed


We have used temperature gradient gel electrophoresis (TGGE) to measure the progress of local denaturation in closed circular topoisomer DNA as a function of temperature and superhelicity (sigma). We describe the versatility of this method as a tool for detecting various conformational modifications of plasmid DNAs. The early melting temperature of a structural transition for any topoisomer is dependent on the value of superhelicity. Supercoiled topo-isomers represent a system of molecules that is sensitive to changes in temperature. We show that the topoisomer with the highest absolute value of superhelicity melts earlier than topoisomers with lower values. Thermal sensitivity of highly supercoiled plasmids could play a biologically important role in regulation of replication and expression in cells under thermal stress. The estimated melting temperature for plasmids with sigma < -0.05 is very significant because these temperatures for early melting are below physiological temperatures.

Download full-text


Available from: Viktor Viglasky
  • Source
    • "Apart from its effect on pDNA stability [12], temperature is a parameter that might significantly influence pDNA interactions with arginine matrix and consequently affect the retention. Thus, a series of chromatographic experiments were designed to show how temperature affects the adsorption of pDNA isoforms to the arginine support. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Arginine chromatography was used to fully separate supercoiled and open circular plasmid DNA (pDNA) isoforms. The results show that the arginine matrix promotes multiple interactions with pDNA, including not only electrostatic and hydrophobic but also biorecognition of nucleotide bases by the arginine ligand. The strong interactions occurring with DNA backbone provide stability, conducting to high effectiveness of arginine support to bind pDNA at low ionic strength. The specific interaction of arginine with sc pDNA could be due to the ability of arginine matrix to be involved in complex interactions that are partly dependent on the conformation of the DNA molecule.
    Full-text · Article · Apr 2008 · Analytical Biochemistry
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A class of simple statistical mechanical models for DNA melting, first proposed by Poland and Scheraga, has been demonstrated to exhibit a first or second order thermodynamic singularity, notwithstanding the intrinsic one-dimensional nature of the problem. In the present paper we consider the case of circular DNA and show that the inclusion of twist elastic energy in the Poland-Scheraga models leads either to suppression of the thermodynamic singularity or to a weak, third order singularity. Such behavior may also be present in linear DNA under mechanical influences that preclude the release of torsional strain.
    Preview · Article · Apr 2002 · Physical Review E
  • [Show abstract] [Hide abstract]
    ABSTRACT: Changes in DNA supercoiling might be essential to generate the response of cellular machinery to temperature stress. The heat-induced structural transition for a topoisomer depends on the value of its specific linking difference. We detect only less negatively supercoiled DNA and an abundance of alternative irregular DNA forms at culture temperatures close to the growth limit of Escherichia coli. We show that the irregular forms are derived from regular plasmid DNAs and their population in the cells is temperature-dependent. Here, we show that it is possible to isolate and characterize individual DNA topoisomers directly from cells without a topoisomerase treatment. Temperature gradient gel electrophoresis (TGGE) and atomic force microscopy (AFM) were used to study the effect of bacteria growth temperature on the distribution of supercoiled DNA and its thermal stability.
    No preview · Article · Oct 2002 · Electrophoresis
Show more