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. 2015 Oct 12:6:8440.
doi: 10.1038/ncomms9440.

Structural diversity of supercoiled DNA

Rossitza N Irobalieva  1   2 , Jonathan M Fogg  2   3   4 , Daniel J Catanese Jr  2 , Thana Sutthibutpong  5 , Muyuan Chen  1   2 , Anna K Barker  3 , Steven J Ludtke  1   2 , Sarah A Harris  5 , Michael F Schmid  1   2 , Wah Chiu  1   2   3 , Lynn Zechiedrich  1   2   3   4
Affiliations

Structural diversity of supercoiled DNA

Rossitza N Irobalieva et al. Nat Commun. .

Erratum in

  • Erratum: Structural diversity of supercoiled DNA.
    Irobalieva RN, Fogg JM, Catanese DJ Jr, Sutthibutpong T, Chen M, Barker AK, Ludtke SJ, Harris SA, Schmid MF, Chiu W, Zechiedrich L. Irobalieva RN, et al. Nat Commun. 2015 Oct 29;6:8851. doi: 10.1038/ncomms9851. Nat Commun. 2015. PMID: 26511109 Free PMC article. No abstract available.

Abstract

By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism. Despite its importance, however, much about supercoiled DNA (positively supercoiled DNA, in particular) remains unknown. Here we use electron cryo-tomography together with biochemical analyses to investigate structures of individual purified DNA minicircle topoisomers with defined degrees of supercoiling. Our results reveal that each topoisomer, negative or positive, adopts a unique and surprisingly wide distribution of three-dimensional conformations. Moreover, we uncover striking differences in how the topoisomers handle torsional stress. As negative supercoiling increases, bases are increasingly exposed. Beyond a sharp supercoiling threshold, we also detect exposed bases in positively supercoiled DNA. Molecular dynamics simulations independently confirm the conformational heterogeneity and provide atomistic insight into the flexibility of supercoiled DNA. Our integrated approach reveals the three-dimensional structures of DNA that are essential for its function.

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Conflict of interest statement

Competing financial interests

J.M.F., D.J.C., and L.Z. are co-inventors on several patents covering the minicircle technology and founders and shareholders in Twister Biotech, Inc.

Figures

Figure 1
Figure 1. Effect of supercoiling on the structure of minicircle DNA
a, Individual 336 bp minicircle topoisomers were isolated and analyzed 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 analyzed). 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-8s a value of 2, figure-8s as a value of 3, etc. 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).
Figure 2
Figure 2. Computational tracing of 336 and 672 bp minicircles
a, Docking of 336 bp traces into the cryo-ET densities of open circles. Traces (purple lines) were generated by docking circular strings of length 336 bp into the density maps. Each trace was then used to isolate the minicircles (grey surfaces) from the cryo-ET density maps. b, Docking of 336 bp traces into the cryo-ET tomograms of writhed minicircles following the same protocol as in a. c, Docking of double-length (672 bp) traces into the cryo-ET tomograms following the same protocol as for 336 bp.
Figure 3
Figure 3. Comparison of electrophoretic mobility and radius of gyration values
a, Left, distance each topoisomer migrated during polyacrylamide gel electrophoresis, measured from the well to the center of the band (Fig. 1a), relative to the migration of the linearized 336 bp minicircle. Data shown are the mean values from three separate gels run under identical conditions. Middle, average radius of gyration values obtained from cryo-ET density maps for each topoisomer (n = 23, 78, 40, 60, 47, 56, and 159 for topoisomers ΔLk = −3, −2, −1, 0, 1,2 and 3 respectively). Right, radius of gyration (averaged over time) in continuum solvent MD simulations for each topoisomer. Error bars for each of the three graphs represent standard deviation values. b, Comparison of cryo-ET data and the equivalent conformations as observed in MD simulations. Examples from negatively supercoiled (ΔLk = −2) and positively supercoiled (ΔLk = +1) topoisomers are shown. MD simulation data are depicted as double-stranded DNA backbone traces.
Figure 4
Figure 4. Effect of supercoiling on DNA base accessibility
a, Minicircle DNA incubated with nuclease Bal-31. Over time, samples were removed, quenched by the addition of stop buffer, and the products analyzed by polyacrylamide gel electrophoresis. Mr: 100 bp DNA ladder, L: linearized 336 bp DNA, N: nicked 336 bp minicircle. b, Graphic representation of the data shown in a. Fitted lines are for visualization purposes only. c, MD simulation of the ΔLk = −3 topoisomer in explicit solvent. Splayed bases were found at a sharp bend of a needle conformation. This may be a potential atomistic explanation for Bal-31 susceptibility of negatively supercoiled topoisomers.
Figure 5
Figure 5. Mapping Bal-31 cleavage
To determine whether Bal-31 cleavage occurs at multiple sites or at a preferred site, the ΔLk = −6 topoisomer was cleaved with Bal-31 and various restriction enzymes. a, Products were separated by agarose gel electrophoresis. Left, (lanes 1–5), control reactions, mc336 (approximately equal mixture of ΔLk = −2 and ΔLk = −3 topoisomers) with combinations of the various restriction enzymes (as indicated) to generate fragments of known DNA lengths. Right, (lanes 6–9), ΔLk = −6 topoisomer cleaved first with Bal-31, followed by a restriction enzyme (as indicated). Mr1: 100 bp DNA ladder, Mr2: Low molecular weight DNA ladder. b, Map of the minicircle sequence showing the positions of the restriction enzymes used, the estimated location of Bal-31 cleavage (with parentheses indicating the range), and the location of the observed base-pair breaking in MD simulation of the ΔLk = −3 topoisomer.
Figure 6
Figure 6. Model for how DNA accommodates supercoiling
Comparison of smooth and sharp bending and the effect of localized denaturation. Images represent a more detailed view of the local structure at the bend. For smooth bending, writhe-mediated bending is regular with bending strain more evenly distributed. Base flipping may generate flexible hinges, allowing DNA to bend more sharply or kink. Alternatively, writhe-mediated sharp bending may lead to disruption of base pairs, even in positively supercoiled DNA. More extensive denaturation may release torsional strain and allow DNA to adopt more open conformations. Denaturation bubbles also provide a flexible joint allowing DNA to kink.
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