Jornal de tecnologia microbiana e bioquímica

Abstrato

DNA Spiral Supercoiling and Intramolecular Topological Interlink

Xitai Huang, Jia Yu, Zhenfeng Zhang and Kou Cao

Escherichia coli chromosome DNA was observed previously to consist of subunits of Archimedean spirallike supercoiling. How do cells build such a DNA structure remains unknown. In the present study, atomic force microscopy (AFM) images showed that supercoiled pBR322 DNA forms a spiral structure upon intercalation with 0.5 μg/ml ethidium bromide (EB), which used to be considered as zero superhelical density. New evidence suggested that a novel topological bond, intramolecular topological interlink (ITL), promotes the DNA spiral formation. Without intercalation, the supercoiled pBR322 DNA displays plectonemical supercoil with uneven distribution of the supercoil density. Similar observation was also made when the DNA was over intercalated by EB (20 μg/ml). The results indicated that ITL functions as a brake to block the helical double strands twisting and comparted circular DNA into different superhelical density domains. When DNA was denatured in alkaline, AFM images showed that the ITL remains constant. As the denatured pBR322 DNA was cut with restriction endonuclease PstI, the digested DNA kept intramolecular interlinks to converge into a centre with two free cut ends. Interlinked intermediates can be observed when natural pBR322 DNA was digested with HindIII and site-specific nickase Nb. Bpu10I. All evidence suggests that ITL is present in pBR322 DNA and causes DNA spiral supercoiling. It was found that the DNA topoisomers with different ITL number run into a ladder of bands in electrophoresis which is distinct from the DNA topoisomers produced by E. coli gyrase forming a smear. In a cell free system containing E. coli cell extract, we demonstrate that topoisomerase IV is required for the production of ITL DNA topoisomers from relaxed cccDNA substrate. Collectively, our data suggest that ITL represents a novel element of DNA topological structure. The DNA spiral supercoiling may be a universal structure present in the cell.