Exploring writhe in supercoiled minicircle DNA

Abstract

Using λ-Int recombination in E. coli, we have generated milligram quantities of supercoiled minicircle DNA. Intramolecular Int recombination was efficient down to lengths ~254 bp. When nicked and religated in the presence of ethidium bromide, 339 bp minicircles adopted at least seven unique topoisomers that presumably correspond to ΔLk ranging from 0 to -6, which we purified individually. We used these minicircles, with unique ΔLk, to address the partition into twist and writhe as a function of ΔLk. Gel electrophoresis and atomic force microscopy revealed progressively higher writhe conformations in the presence of 10 mM CaCl(2) or MgCl(2). From simplistic calculations of the bending and twisting energies, we predict the elastic free energy of supercoiling for these minicircles to be lower than if the supercoiling was partitioned mainly into twist. The predicted writhe corresponds closely with that which we observed experimentally in the presence of divalent metal ions. However, in the absence of divalent metal ions only limited writhe was observed, demonstrating the importance of electrostatic effects on DNA structure, when the screening of charges on the DNA is weak. This study represents a unique insight into the supercoiling of minicircle DNA, with implications for DNA structure in general.

Figure 1

Generation of minicircle DNA by integrase-mediated site-specific recombination. Site-specific recombination between attB and attP sites, directly orientated on the same plasmid, exchanges the sequences denoted by the arrowheads, to form two catenated DNA circles. These catenanes are separated by cleavage of the large circle using BamHI, which releases the small supercoiled minicircle.

Figure 2

Religation of minicircle DNA in the presence of ethidium bromide. Ligation was carried out with increasing concentrations of ethidium bromide. The intercalator was extracted with butanol, and the products displayed by polyacrylamide gel electrophoresis, in the presence of 10 mM CaCl₂. No ethidium bromide was added to the gel.

Figure 3

Electrophoresis of minicircle DNA in the presence of (A) 10 mM CaCl₂, or (B) 2 mM EDTA. M: 100 bp DNA ladder (NEB); G: minicircle from in vivo (integrase) reaction after gel filtration; P: after preparative gel electrophoresis; L: minicircle linearized by NdeI; N: minicircle nicked by Nt.BbvC I; lanes 6–12: topoisomers of minicircle, generated by religation of nicked minicircle and purified by preparative gel electrophoresis.

Figure 4

Analysis of DNA minicircles by atomic force microscopy (AFM). (A) Individual molecules were categorized into one of the five classes, OPEN, BEAN, ONE, TWO+ or ROD. Examples of each of the classes and total number of molecules in each class (over all topoisomers) are shown. (B) Relative proportions of each class as a function of ΔLk. The error bars were calculated according to the equation $\sqrt{p(1-p)/n}$, where p is the population and n is the number of molecules in the class (for each topoisomer).

Figure 5

Integrase-mediated recombination efficiency as a function of distance between attB and attP sites. (A) Products of integrase recombination separated by agarose gel electrophoresis. Lane 1: 100 bp DNA ladder (NEB); lane 2: 1 kb DNA ladder (NEB); lanes 4–11: products of integrase recombination for various spacings between the attB and attP sites. (B) Efficiency of intramolecular recombination as a function of distance between attB and attP sites. Inset: efficiency of intramolecular recombination over the range 200–500 bp.

Figure 6

Generation of dimers by integrase recombination. Site-specific recombination between attB and attP sites on different plasmids leads to the formation of a dimer plasmid. Further recombination between the remaining attB and attP sites leads to catenane formation. Cleavage of the large circle by BamHI releases the minicircle DNA which is twice the size of minicircle generated by recombination between sites on the same plasmid (Figure 1).

Figure 7

Predicted free energies for different values of writhe for each of the topoisomers of the 339 bp minicircle. Free energies were calculated as described in the text. Diamonds indicate energy minima for each topoisomer. Lowest predicted free energies occurred for ΔWr of 0, 0.3, 0.9, 1.4, 2.0, 2.5 and 3.1, for topoisomers ΔLk = 0, −1, −2, −3, −4, −5 and −6 respectively.