Single-molecule micromanipulation studies of methylated DNA

Abstract

Cytosine methylated at the five-carbon position is the most widely studied reversible DNA modification. Prior findings indicate that methylation can alter mechanical properties. However, those findings were qualitative and sometimes contradictory, leaving many aspects unclear. By applying single-molecule magnetic force spectroscopy techniques allowing for direct manipulation and dynamic observation of DNA mechanics and mechanically driven strand separation, we investigated how CpG and non-CpG cytosine methylation affects DNA micromechanical properties. We quantitatively characterized DNA stiffness using persistence length measurements from force-extension curves in the nanoscale length regime and demonstrated that cytosine methylation results in longer contour length and increased DNA flexibility (i.e., decreased persistence length). In addition, we observed the preferential formation of plectonemes over unwound single-stranded "bubbles" of DNA under physiologically relevant stretching forces and supercoiling densities. The flexibility and high structural stability of methylated DNA is likely to have significant consequences on the recruitment of proteins recognizing cytosine methylation and DNA packaging.

Figure 1

Verification of CpG methylation. (A). BstUI restriction sites and fragment sizes. (B). CpG methylation of ∼6 kb PCR-created construct was carried out with M.SssI, and the extent of methylation was checked by digestion with methylation-sensitive restriction enzyme BstUI. (C) BstUI digestion of the ∼30% PCR-methylated construct produces a broad range of DNA fragment sizes.

Figure 2

Elastic properties of control and PCR-methylated dsDNA. (A). The force-extension curves of control (black circles) and methylated (gray circles) DNA. (B). Elastic parameters obtained from the force-extension curves (the number of experiments n for each construct is indicated).

Figure 3

Extension rotation and melting curves for single-DNA stretching and winding (+σ)/unwinding (−σ) measured using magnetic tweezers. (A) Rotation curves. Control (empty circles; dashed line) and methylated (filled circles; solid line) ∼6 kb DNA constructs derived from pFOS1 by PCR (see Materials and methods) held at constant force 0.6 pN at various superhelical densities σ. All data points are mean values of DNA extension that are collected within a 40-s window, and corresponding error bars are the SD values of the DNA extension averaging; lines represent the best fit of polynomial regression. (B) Melting curves for control (empty circles; dashed line) and methylated (filled circles; solid line) DNA. Difference in extension (ΔZ = Z(−0.06)−Z(+0.06)) at superhelical density σ ± 0.06 as a function of applied stretching force is shown. Dotted and solid vertical lines represent melting forces for control, or methylated DNA. Results are representative of three independent experiments.