Figure 1: Effect of supercoiling on the structure of minicircle DNA.
(a) Individual 336 bp minicircle topoisomers were isolated and analysed by polyacrylamide gel electrophoresis in the presence of 10 mM CaCl2. Mr: 100 bp DNA ladder, L: minicircle linearized by EcoRV, N: minicircle nicked by Nb.BbvCI. (b) Projections of cryo-ET subtomograms of hydrated 336 bp DNA minicircles of the Lk=34 topoisomer. (c) Commonly observed shapes were open circle, open figure-8, figure-8, racquet, handcuffs, needle, and rod, each of which are shown in orthogonal views. (d) Other shapes observed, especially in the more highly supercoiled topoisomers. (e) Shape frequency distribution plot for each topoisomer population (n=number of minicircles analysed). A weighted average for each topoisomer, approximating the average degree of compactness, is denoted by the black triangle. The weighted average was calculated by assigning each conformation a value that increased in line with compactness. Open circles were given a value of 1, open figure-8 s a value of 2, figure-8 s as a value of 3, and so on. The relative fraction of each was subsequently used to determine the average degree of compactness. Lk, ΔLk and superhelical density (σ) for each topoisomer are shown (see Supplementary Note 1).
