Supercoiling DNA Locates Mismatches

Abstract

We present a method of detecting sequence defects by supercoiling DNA with magnetic tweezers. The method is sensitive to a single mismatched base pair in a DNA sequence of several thousand base pairs. We systematically compare DNA molecules with 0 to 16 adjacent mismatches at 1 M monovalent salt and 3.6 pN force and show that under these conditions, a single plectoneme forms and is stably pinned at the defect. We use these measurements to estimate the energy and degree of end-loop kinking at defects. From this, we calculate the relative probability of plectoneme pinning at the mismatch under physiologically relevant conditions. Based on this estimate, we propose that DNA supercoiling could contribute to mismatch and damage sensing in vivo.

Fig. 1

Method of locating a sequence defect in a single DNA molecule by supercoiling. We use magnetic tweezers to measure DNA extension at constant force while increasing the excess linking number Lk through applied turns of the tethered magnetic bead. For an asymmetrically positioned defect (red dot), two buckling transitions are observed. The first transition, at Lk^† and critical torque Γ_c, causes the torque to abruptly drop by an amount ΔΓ and produces a pinned plectoneme with the defect at its tip. Surface encounter of the initial plectoneme prevents it from lengthening and causes re-buckling of the DNA at a torque larger than Γ_c. The torque at Lk* is assumed equal to the plateau torque Γ* that is independent of defects and determined from |∂X/∂Lk| after re-buckling [11, 12].

Fig. 2

Data from 6 kb DNA with n = 0 to 16 adjacent mismatches measured at 3.6 pN force and 1 M salt. (a) Example time-series recorded near linking number Lk = Lk^† of fluctuations between unbuckled and buckled states, which differ in extension by ΔX. (b) Data of extension vs excess linking number [14]. For n = 0, only single buckling is observed (arrow); for n > 0 re-buckling of intact DNA is observed (arrows) due to the surface encounter of the defect-pinned plectoneme. (c) The change in DNA extension upon buckling decreases quadratically with defect size, n. The dashed curve is the fit to ΔX_n = a_X − c_X·n², with a_X = 88.3 ± 0.3 nm and c_X = 2.94 ± 0.08 nm. (d) The linking number change (Lk^† − Lk* [12], left axis) also decreases as a_Lk − c_Lk · n², with best-fit parameters a_Lk = 3.7 ± 0.1 and c_Lk = 0.17 ± 0.01. This provides an estimate of the critical buckling torque (right axis); see text.

Fig. 3

Experimental salt-force phase diagram of re-buckling for n = 2. Re-buckling requires high force and ionic screening. The shaded region represents conditions in which re-buckling occurred in greater than 50% of repeated measurements (+ points; see Figs. S2 and S3 in Supporting Information). The data in Fig. 2 were collected at 3.6 pN and 1 M salt (circled).

Fig. 4

Model of end-loop kinking to estimate plectoneme pinning probabilities. (a) Energy scaling factor ∊ calculated for a planar end-loop as a function of kinking angle. (The interior apex angle and kinking angle are supplementary.) (b) Estimate of ∊ with defect size based on data in Fig. 2c. (c) Estimated enhancement, $\langle m_{\in }^{†}\rangle /\left(\langle m\rangle /N\right)$, of plectoneme occupancy at the defect relative to any other position along a N = 10 kb topological domain of DNA at supercoiling density 0.05 and 0.2 M monovalent salt [4]. Points indicate independent calculations at each force and kinked end-loop energy.