Studies On DNA Topology And DNA Topoisomerases

DNA is supercoiled in cells. DNA supercoiling belongs to the research field of topology. DNA advanced structure is also called DNA topological structure or DNA topology. DNA supercoiling is essential for various cellular process and DNA topoisomerase are responsible for controlling the superhelicity of DNA.To study the alkali denaturation of supercoiled DNA, plasmid pBR322 was treated with gradient concentrations of NaOH solution. The results of gel electrophoresis showed that the alkali denaturation of the supercoiled DNA occurred in a narrow range of pH value (12.88 to 12.90). The alkali-denatured supercoiled DNA ran, as a sharp band, faster than the supercoiled DNA. Three kinds of supercoiled plasmid DNA were denatured by NaOH, and then visualized by atomic force microscopy. Compared with the supercoiled DNA, the atomic force microscopy images of the alkali-denatured supercoiled DNA showed rough surface with many kinks, bulges on double strands with inhomogeneous double duplex diameters. The perimeters of the denatured DNA were shortened. All evidence suggested that the alkali-denatured supercoiled DNA had a stable conformation with unregistered, topologically constrained double strands and intrastrand secondary structure. Theses structural alterations are responsible for the special behavior of DNA IV in agarose gel electrophoresis.Escherichia coli chromosome DNA was observed previously to consist of subunits of Archimedean spiral-like supercoiling. How cells build such a DNA structure remains unknown. In this study, we show that intramolecular topological interlink (ITL), a novel topological bond, is the essential structural element that connects DNA double strands at different parts of the molecule by cross interlinking. It comparts DNA into different topological domains, which support the spiral supercoiling in the plasmid pBR322 DNA isolated from E. coli. The diameter of the pBR322 DNA topoisomers examined by atomic force microscopy (AFM) was found to be reversely proportional to the ITL numbering. It is the DNA topoisomers with different ITL number running into a ladder of bands in agarose gel electrophoresis which is different from the DNA topoisomers produced by E. coli gyrase alone running in a smear. The results suggest that ITL is independent of the well-known DNA topological equation:Lk=Tw+Wr. Site-specific single strand DNA nicking was able to release ITL of supercoiled DNA in a time-consuming course. In a cell free system containing E. coli cell extract, E. coli topoisomerase I, gyrase, topoisomerase III and topoisomerase IV create different DNA topological products with relaxed covalent closed circular DNA (cccDNA) substrate. However, it is the mixture of topoisomerase IV catalyzing production of the ladder of ITL DNA topoisomers only. The results indicate that topoisomerase IV is responsible to intramolecular topological interlinking with gyrase cooperation. Collectively, our data suggest that ITL is the essential structural element for DNA spiral supercoiling. The DNA spiral supercoiling is superior to plectonemical supercoiling and solenoidal supercoiling for chromosome DNA compaction and accessibility. Our finding suggests that DNA spiral supercoiling may be a universal structure present in the cell.Atomic force microscopy (AFM) has been increasingly used in the investigation of DNA structure. In previous studies, it was short of appropriate rules condition in depth analysis and objective evaluation for the DNA image quality. In this study, we established an evaluation system for image quality of DNA fine structure by AFM and three parameters were applied for image quality evaluation of AFM. According to this system, the appropriate conditions for DNA topological structure by AFM were confirmed by the evaluation of three key factors:the concentration of DNA, the concentration of magnesium ion and the relative humidity.