Stretched and overwound DNA forms a Pauling-like structure with exposed bases

Abstract

We investigate structural transitions within a single stretched and supercoiled DNA molecule. With negative supercoiling, for a stretching force >0.3 pN, we observe the coexistence of B-DNA and denatured DNA from sigma approximately -0.015 down to sigma = -1. Surprisingly, for positively supercoiled DNA (sigma > +0.037) stretched by 3 pN, we observe a similar coexistence of B-DNA and a new, highly twisted structure. Experimental data and molecular modeling suggest that this structure has approximately 2.62 bases per turn and an extension 75% larger than B-DNA. This structure has tightly interwound phosphate backbones and exposed bases in common with Pauling's early DNA structure [Pauling, L. & Corey, R. B. (1953), Proc. Natl. Acad. Sci. USA 39, 84-97] and an unusual structure proposed for the Pf1 bacteriophage [Liu, D. J. & Day, L. A. (1994) Science 265, 671-674].

Figure 1

(Upper) Schematic representation of the experiment. A DIG/biotine end-labeled DNA molecule (≈5 μm long) was attached at one end to a glass surface by DIG/anti-DIG bonds and at the other to a 4.5-μm magnetic bead via streptavidine/biotin links. By varying the distance between the sample and the permanent magnets, the stretching force was controlled whereas rotating the magnets induced DNA supercoiling. The sample was placed on an inverted microscope and was viewed with a ×63 objective. Real-time video analysis of the bead’s image provided a measure of the molecule’s extension and the force via its Brownian fluctuations. Greater forces led to more restricted fluctuations. (Lower) Extension l vs. σ for various forces. Below a critical force F_c− (=0.3 pN in 10 mM PB), the curve was symmetric: for both positive and negative σ, the molecule shortened as it writhed and formed supercoils (plectonemes). Pulling on the molecule increased the torque by reducing the writhe. For forces F_c− < F < F_c+ (=3 pN in 10 mM PB), the molecule shortened only for positive σ. For negative σ, the decrease in twist was adsorbed by the formation of dDNA appearing below σ_c− = −0.015. At forces F > F_c+, the molecule no longer shortened on winding. dDNA appeared for σ < −0.15 and P-DNA when σ > +0.037. These structural transitions allowed for a stabilization of the torque.

Figure 2

Experimental evidence for the coexistence in 10 mM PB of B-DNA and denatured DNA at negative supercoiling: −1 < σ < 0. (A) Force (F) vs. extension (l) curves of a single DNA molecule obtained at different degrees of supercoiling. At F = F_c− = 0.3 pN, the extension of the molecule changed dramatically pointing to the presence of a structural transition in DNA: denatured DNA appeared. (B) The data from A were rescaled such that l_d(F) = (σ_d/σ)[l(F,σ) − l(F,0)] + l(F,0) (see Eq. 1). All data collapsed on a single curve, validating our hypothesis that stretched, unwound DNA separates into pure B-DNA and dDNA phases. l_d(F) is the extension vs. force curve for denatured DNA. Notice that it is not the simple superposition of two parallel and noninteracting single-stranded DNAs (4). (C) The fraction φ_d of dDNA is plotted as a function of the number of turns. φ_d was obtained by a mean square fit of the force curve measured at a given σ and was expressed as a linear combination of the force curves obtained, respectively, at σ = 0 and σ = −1. As expected from Eq. 1, φ_d = −σ and goes from 0 to 1. Beyond this value, the points depart from linearity.

Figure 3

Detection of unpaired bases after incubation of a stretched and twisted DNA in glyoxal. Extension vs. torsion curves were obtained at a constant stretching force F between 0.2 and 0.3 pN. When no glyoxal was used (control experiment), the extension displayed a pronounced maximum at σ = 0. The formation of plectonemes on positive or negative twisting led to a rapid shortening of the molecule. When a stretched molecule unwound by n_inc = 200 turns was incubated in glyoxal, the subsequent l vs. σ curve (obtained after washing out the glyoxal) displayed a clear plateau. Its width (≈180 turns) was roughly equal to n_inc, which indicates that, beyond a threshold (≈20 turns, i.e., σ ≈ −0.015), every turn applied to a stretched and unwound molecule induced the melting of one turn of the double helix (10.5 base pairs). Molecular modeling suggests that the structure appearing in overwound DNA (P-DNA) also should have unpaired bases. Incubation in glyoxal of a different stretched and overwound molecule confirmed this prediction. After washing out the glyoxal, the l vs. σ curve displays the same plateau as for the underwound case. However, note that, to get the same plateau required three times more turns being applied to the molecule than in the former case. This is consistent with the measurements of σ_d = −1 and σ_p = +3.

Figure 4

Mechanical characterization of P-DNA. (A) Elasticity curves showing two sharp transitions at 3 and 25 pN. The first transition (not shown for all curves) is associated with the disappearance of plectonemes in B-DNA and the formation of P-DNA. The second transition, showing hysteresis, is attributed to the disappearance of plectonemes in the P-DNA sub-phase. Note that, because of the possibility of stabilizing interactions between exposed bases, these plectonemes should be more stable than with B-DNA. The existence of these plectonemic structures also might explain the shortening of the molecule at relatively low forces 3 pN < F < 10 pN. At high force, these curves show that P-DNA is actually longer than B-DNA. (B) A detailed view of the curves from A for decreasing forces (each curve corresponds to σ_i = i × 0.343). (C) Rescaling, following Eq. 1, enabled all of the curves shown in B to be collapsed to a single curve l_p(F) that describes the extension vs. force behavior of a pure P-DNA. The full line is a fit to the model for P-DNA (Eq. 2) with l_p,0 = 1.75, ξ_p = 19 nm, and ɛ = 0.12 k_BT/nm (see text for details).

Figure 5

Structure of P-DNA deduced from molecular modeling. Space-filling models of a (dG)₁₈⋅(dC)₁₈ fragment in B-DNA (Left) and P-DNA (Right) conformations. The backbones are colored purple, and the bases are colored blue (guanine) and yellow (cytosine). The anionic oxygens of the phosphate groups are shown in red. These models were created with the jumna program (11, 12) by imposing twisting constraints on helically symmetric DNAs with regular repeating base sequences.

Figure 6

Theoretical modeling of the conformational energy of (dG)₁₈⋅(dC)₁₈. (A) Energy as a function of the twist per base pair. Base pair breaking and expulsion occurred at ≈70° (solid line) coupled to a sudden relaxation of the strain energy built up in the backbones. The discontinuity at 130° corresponds to a rearrangement of the backbones so that the phosphate anionic oxygens point outwards. This introduced C3′-endo puckering in the cytidine strand. When Watson–Crick hydrogen bonding was maintained with distance constraints the energy rose rapidly beyond a twist of 70° (dotted line). (B) Energy as a function of the rise per bp for overtwisted DNA (twist constrained at 138°). The rise could be varied between ≈4 and 6 Å (80% of the maximum extension of a single strand of DNA) with little energy change. The discontinuity at 5.5 Å corresponds to the reintroduction of a C2′-endo sugar for the cytidines, allowing an increase in the intrastrand P-P distance.