Abstract
Cytosine methylation plays an essential role in many biological processes, such as nucleosome inactivation and regulation of gene expression. The modulation of DNA mechanics may be one of the regulatory mechanisms influenced by cytosine methylation. However, it remains unclear how methylation influences DNA mechanics. Here, we show that methylation has contrasting effects on the bending property of dsDNA depending on DNA curvature. We directly applied bending force on 30 base pairs of dsDNA using a D-shaped DNA nanostructure and measured the degree of bending using single-molecule fluorescence resonance energy transfer without surface immobilization. When dsDNA is weakly bent, methylation increases the stiffness of dsDNA. The stiffness of dsDNA increased by approximately 8% with a single methylation site for 30 bp dsDNA. When dsDNA is highly bent by a strong force, it forms a kink, i.e., a sharp bending of dsDNA. Under strong bending, methylation destabilizes the non-kink form compared with the kink form, which makes dsDNA near the kink region apparently more bendable. However, if the kink region is methylated, the kink form is destabilized, and dsDNA becomes stiffer. As a result, methylation increases the stiffness of weakly bent dsDNA and concurrently can promote kink formation, which may stabilize the nucleosome structure. Our results provide new insight into the effect of methylation, showing that cytosine methylation has opposite effects on DNA mechanics depending on its curvature and methylation location.