Exploring writhe in supercoiled minicircle DNA

Abstract

Using λ-Int recombination in E. coli, we have generated milligram quantities of supercoiledminicircle DNA. Intramolecular Int recombination was efficient down to lengths∼254 bp. When nicked and religated in the presence of ethidium bromide, 339 bp minicirclesadopted at least seven unique topoisomers that presumably correspond toΔLk rangingfrom 0 to −6, which we purified individually. We used these minicircles, with uniqueΔLk, to address the partition into twist and writhe as a function ofΔLk. Gel electrophoresis and atomic force microscopy revealedprogressively higher writhe conformations in the presence of 10 mMCaCl2 orMgCl2. From simplistic calculations of the bending and twisting energies, we predict the elasticfree energy of supercoiling for these minicircles to be lower than if the supercoiling waspartitioned mainly into twist. The predicted writhe corresponds closely with that which weobserved experimentally in the presence of divalent metal ions. However, in theabsence of divalent metal ions only limited writhe was observed, demonstratingthe importance of electrostatic effects on DNA structure, when the screening ofcharges on the DNA is weak. This study represents a unique insight into thesupercoiling of minicircle DNA, with implications for DNA structure in general.