336 bp minicircles accommodate a wide range of supercoiling

Minicircles containing 336 bp were selected for this study because they are representative of the supercoiled DNA loops found in nature15,16,17,18. Furthermore, these minicircles are small enough to allow isolation of suitable amounts of individual topoisomers (Fig. 1a), yet large enough to yield an ample spread of ten unique topoisomers (Supplementary Fig. 1). In its relaxed state, the two strands of a 336 bp DNA circle wrap around each other 32 times. This number is known as the linking number (Lk). The other topoisomers deviate (ΔLk) from the Lk of the relaxed topoisomer (Lk=32, ΔLk=0) in steps of 1. We generated and isolated six different negatively supercoiled minicircle topoisomers (Lk=31 through 26, ΔLk=−1 through −6), three different positively supercoiled topoisomers (Lk=33 through 35, ΔLk=+1 through +3), relaxed (Lk=32, ΔLk=0), nicked and linearized minicircles (Fig. 1a, Supplementary Table 1).

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 CaCl 2 . 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). Full size image

All nine of the supercoiled minicircles were subjected to and relaxed by human topoisomerase IIα (htopoIIα) (Supplementary Fig. 2). This relaxation demonstrated the biological activity of the minicircles, confirmed their Lk designations, and verified that there was no cross-contamination among topoisomers. Because htopoIIα relaxes in characteristic steps of two Lk, all of the minicircles with even Lk relaxed to ΔLk=0. The odd-numbered Lk topoisomers relaxed to a mixture of ΔLk=−1 and ΔLk=+1.

Wide variety of minicircle DNA conformations

Electron cryo-tomography (cryo-ET) was used to obtain three-dimensional (3D) structures of the different minicircles embedded in vitreous ice. Before freezing, the purified topoisomers were either incubated on ice or at room temperature for at least 15 min in an attempt to allow the DNA to reach conformational equilibrium. Specimen vitrification19 occurs at a rate of freezing (106 °C per second) that should be fast enough to preclude temperature-dependent structural alterations20. Projection images were obtained with an electron microscope by incrementally tilting the specimen stage. These images were subsequently reconstructed into 3D volumes (tomograms) containing the minicircles (Supplementary Movie 1). Thus, these data represent snapshots of the DNA molecules at the instant of freezing. We computationally generated traces of 336-bp minicircle DNA backbones and fitted these to the observed densities in the tomograms, confirming that each subvolume represented a single 336 bp minicircle (Fig. 2a,b, Methods). In addition, as a control, we visualized double-length minicircles (672 bp) and computationally fitted a 672 bp backbone to the observed densities, confirming their length and thus providing additional validation of the approach (Fig. 2c).

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 for the open circles. (c) Docking of double-length (672 bp) traces into the cryo-ET tomograms following the same protocol as for 336 bp. Full size image

A broad mixture of 3D conformations was observed for each purified 336-bp minicircle topoisomer (Fig. 1b,e). Given the rigidity of DNA at this short length21, it is remarkable that the DNA was able to contort into such a wide variety of conformations. The heterogeneity observed indicates that the DNA structure is in dynamic equilibrium, driven by both the torsional stress of supercoiling and by Brownian motion13. These results illustrate the power of cryo-ET to visualize individual molecules in solution and capture their conformational variability. Furthermore, cryo-ET data contained sufficient detail to visualize that, as expected, only right-handed crossovers were observed in negatively supercoiled DNA and only left-handed crossovers were observed in positively supercoiled DNA.

Most of the observed minicircle conformations could be classified into the following empirical categories, in order of increasing compaction: open circle, open figure-8, figure-8, racquet, handcuffs, needle, and rod (Fig. 1b,c). Examples of the handcuffs and needle conformations are shown in Supplementary Movie 2 and 3. It is interesting to note that minicircles in topologically distinct topoisomers sometimes adopted the same general conformation (for example, rods found in the ΔLk=−1 and ΔLk=−6 topoisomers appear similar). In these cases, DNA supercoiling must be accommodated in different ways that result in the same general 3D shape. Some minicircles, especially those with larger ΔLk (either negative or positive) adopted alternative shapes (‘other’ in Fig. 1d).

Deviations from relaxed DNA may be manifested as changes in twist, the coiling of the DNA about the helical axis, or writhe, the coiling of the double helices about each other. Changes in twist result in torsional strain whereas changes in writhe result in bending strain. Because we observed multiple DNA conformations in supercoiled DNA—ranging from open to highly writhed—different degrees of twist and writhe must simultaneously exist within the same topoisomer population. DNA bending, and thus writhe, is thought to be more difficult to accommodate for short DNA lengths. Minicircles twice the length (672 bp) with equivalent supercoiling (ΔLk=−4) to the ΔLk=−2, 336 bp minicircle all appeared highly writhed (Fig. 2c). In comparison the ΔLk=−2, 336 bp topoisomer displayed both open and writhed conformations (Fig. 1e). Conversely, smaller minicircles of 94–158 bp have no appreciable writhe11,12,13. Minicircles (336 bp) are, therefore, an ideal size for exploring the relationship between twist and writhe.

As mentioned above, linear DNA is rigid in the aforementioned lengths. Theory therefore predicts that very small circles should be perfectly round. Significant deviations from a perfect circle will require non-uniform distribution of bending along the DNA length. Very small circles already have considerable bending strain. Localized variations in bending should therefore be energetically unfavourable. Ellipticity was measured previously in 94–158 bp DNA minicircles and averaged between 1.1 and 1.5 (refs 11, 12, 13). Increased ellipticity was attributed to the appearance of hyperflexible kinks within the DNA13. Thus ellipticity is one measure of conformational variability. To compare our data to those previous studies, we measured ellipticity in a subset of our observed open circles. Significant numbers of minicircles with an open circle shape were found in the following topoisomer populations: ΔLk=−2, 0, +1, +2, +3, and nicked. The measured ellipticity values ranged from 1.1 to 2.6. This larger deviation from circularity is attributable to the longer length of our 336 bp minicircles. We also observed potential differences with supercoiling in the dimensions of the open circles (Supplementary Fig. 3).

The conformations adopted by each topoisomer are shown as frequency distribution plots (Fig. 1e). A weighted average of the shape frequency distribution (denoted by arrowheads on each plot) approximates the average degree of compactness. Similarly, electrophoretic mobility provides a measure of relative compaction22. By both measures increased negative or positive supercoiling leads to a shift in the distribution towards more compact shapes. To provide a more quantitative measure of compactness, radius of gyration values were measured from the cryo-ET density maps (Fig. 3a, middle). These values were consistent with the electrophoretic mobilities (Fig. 3a, left).

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 centre of the band (Fig. 1 a), 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 s.d. 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. Full size image

Molecular dynamics simulations provide atomistic insight

To further understand the conformational fluctuations of the topoisomers, we performed molecular dynamics (MD) simulations (Supplementary Movie 4 and 5; Supplementary Tables 2 and 3). Generalized Born continuum solvent simulations, which, in the absence of viscous damping enable rapid configurational sampling in a relatively short computational time, revealed a wide variety of conformations for each topoisomer. The simulation results suggest that each supercoiled topoisomer undergoes large fluctuations in writhe (Supplementary Movie 4 and Supplementary Fig. 4) and, hence, in the level of compaction. The radius of gyration values, averaged over the MD simulations, showed the same trends with changing superhelical density as the gel electrophoretic mobility and the radius of gyration values extracted from the cryo-ET data (Fig. 3a). Conformations that were observed in the cryo-ET data were also observed in the MD simulations (Fig. 3b), providing insight into how conformations may interchange. The consistency between structural and computational results established confidence for the observation of simultaneous co-existence of multiple conformations of each topoisomer.

Across all levels of supercoiling, differences were observed for negative versus positive ΔLk. Although the ΔLk=−1 and ΔLk=+1 topoisomers might be expected to migrate with similar electrophoretic mobility, ΔLk=−1 migrated much faster on the gel (Fig. 1a). In addition, cryo-ET data revealed that the ΔLk=−1 topoisomer adopted a spread of conformations that included predominantly compact forms (Fig. 1e). The ΔLk=+1 adopted mostly open conformations, similar to the nicked and relaxed minicircles (Fig. 1e). The presence of a small fraction (∼10%) of compact forms may explain why, on average, the ΔLk=+1 topoisomer migrated slightly farther on the gel than the nicked or relaxed minicircles.

Extending the comparison to ΔLk=−2 and ΔLk=+2, we observed broad distributions of conformations, such that every shape category described in Fig. 1c was seen. Overall, the distribution trends for the ΔLk=−2 and ΔLk=+2 were similar. One noteworthy difference, however, was in the relative proportions of the figure-8 and racquet conformations. Racquets were observed ∼3-fold more frequently than figure-8 s for the ΔLk=−2 topoisomer; figure-8 s were ∼5-fold more frequent than racquets for ΔLk=+2. This observation implied structural differences between the two topoisomers, which we verified biochemically as described below.

Further underwinding (ΔLk=−3, −4 and −6) resulted in an additional shift in the distribution toward more compact shapes observed in the cryo-ET data and a concomitant increase in electrophoretic mobility. The increase in electrophoretic mobility between consecutive topoisomers was more pronounced for positive than for negative supercoiling (Fig. 1a). A similar trend was observed for the shift in the conformational distribution between consecutive topoisomers (Fig. 1e). For ΔLk=+3, 39% of the minicircles adopted unusual shapes (‘other,’ Fig. 1d), which were not observed in the ΔLk=+2 population (Fig. 1e), indicating that a sharp structural transition occurs between these two topoisomers.

Probing for base-pair disruptions and localized denaturation

Many of the observed 3D conformations appear to contain sharply bent or kinked DNA. One way that sharp bending may be facilitated is through localized distortions and disruptions of the helix. To probe for and quantify such helix disruptions, we used nuclease Bal-31. Bal-31 has endonuclease function on exposed, unpaired DNA bases (for example, kinks, nicks, gaps, single-stranded regions and B–Z junctions)23,24. All underwound topoisomers and the most overwound ΔLk=+3 topoisomer had some exposed, unpaired bases, as measured by their Bal-31 sensitivity (Fig. 4a). The negatively supercoiled ΔLk=−2, −3, −4 and −6 topoisomers were linearized within the first minute of Bal-31 incubation and subsequently degraded (Fig. 4b). The ΔLk=−1 topoisomer was cleaved at a much slower rate, requiring 20 min to degrade (Fig. 4b). Consistent with their Bal-31 sensitivity, the electrophoretic mobility of the negatively supercoiled topoisomers shifted when incubated with glyoxal, a small molecule that traps exposed bases (Supplementary Fig. 5). The relaxed (ΔLk=0) and ΔLk=+1 topoisomers resisted Bal-31 digestion, and the ΔLk=+2 topoisomer was a very poor substrate (Fig. 4b). Considering that positive supercoiling should inhibit strand separation, it was surprising that the ΔLk=+3 topoisomer was efficiently cleaved by Bal-31 (Fig. 4a) and this topoisomer was almost completely degraded within an hour (Fig. 4b). Single molecule manipulation studies previously uncovered a structural variation of DNA when it was extremely overwound25. The researchers proposed that overwinding may result in an inside-out DNA conformation with the backbones wrapped around each other on the inside and unpaired bases on the outside of the helix. Chemical probing confirmed the presence of unpaired bases. This DNA conformation, named Pauling DNA (P-DNA), was only detected when the DNA was under high tension and writhe was suppressed25. The Bal-31 sensitivity of ΔLk=+3 coincides with the dramatic structural changes observed by cryo-ET for this topoisomer (Fig. 1e). Although the presence of exposed bases may imply P-DNA, there are alternative explanations for Bal-31 sensitivity. A probable explanation for the exposed bases is denaturation resulting from sharp bending. Indeed, sharp bending is a feature of the highly writhed conformations of this topoisomer (see below).

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 analysed 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. Full size image

To provide an atomistic interpretation of the cryo-ET density for each topoisomer and to explore how localized distortions may alter the structural properties of DNA, we performed explicitly solvated MD simulations (Supplementary Movie 6–9). Because the inclusion of solvent molecules in the calculations slows down configurational sampling, these simulations were performed starting from conformers most commonly observed in the continuum solvent MD. The explicit solvent simulations allowed increased conformational diversity and could be directly fitted to the cryo-ET density maps (Fig. 3b and Supplementary Movie 10–13). Whereas restraints were imposed on the hydrogen bond interactions between complementary base pairs in the simulations in implicit solvent, these restrictions were removed for simulations in explicit solvent. This removal of restraints allowed single-stranded regions to form within the minicircles under sufficiently high levels of torsional or bending stress. Accordingly, in simulations of the ΔLk=−3 topoisomer, a kink emerged at the apex of a highly writhed minicircle, where the hydrogen bonding between complementary base pairs was disrupted (Fig. 4c). Similar base-pair disruptions observed by MD simulations have been reported for bent DNA26,27 and for underwound linear DNA28. These computational results are consistent with the experimental observation that the ΔLk=−3 topoisomer is under a high degree of torsional strain, but additionally predicts that base-pair separation may occur at bent apices. Vologodskii and co-workers found that very small DNA minicircles (64–65 bp) were susceptible to Bal-31 cleavage, which was attributed to kinking resulting from the inherent bending strain29. Base-pair disruption has been observed in MD simulations of positively supercoiled DNA, either because of the formation of P-DNA28 or because of sharp bending in very small DNA circles27.

The propensity for base-pair separation depends on DNA sequence30,31. We used the WebSIDD algorithm32 to predict base-pair separation in our 336 bp minicircle, which identified a short region within attR to have a higher probability of duplex destabilization. Because it was very susceptible to Bal-31, the ΔLk=−6 topoisomer was probed. In contrast to the prediction, Bal-31 predominantly cleaved ∼180° away from the anticipated destabilization sequence (Fig. 5). Increased flexibility brought about by localized denaturation at the Bal-31-susceptible site should enable the DNA to be sharply bent. The majority of conformations observed for the ΔLk=−6 topoisomer were sharply bent or kinked (for example, racquets and rods). Kinking at this first site may induce kinking at the site diametrically opposite. Similar cooperative effects between distant sites were seen by Stasiak and co-workers, who observed sequential, cooperative kinking in torsionally strained minicircles at sites located ∼180° apart along the DNA circumference13. An additional influence of the location of Bal-31-susceptible sites may be sequence-dependent DNA flexibility and curvature. Sequences with increased flexibility and/or curvature are preferentially localized to superhelical apices33 where the DNA is most sharply bent. A short region in attR is known to have modest intrinsic curvature34,35. This curvature may be preferentially located at a superhelical apex36, thereby positioning the sequence located ∼180° apart along the DNA circumference at the diametrically opposite superhelical apex. Localized denaturation may be necessary to accommodate the sharp bending at the apex, thereby generating a Bal-31-susceptible site. The kink defect observed by MD at a bent apex (Fig. 4c) is relatively close to the Bal-31 cleavage site (Fig. 5b), consistent with the hypothesis that Bal-31-susceptible sites are localized to the superhelical apices.