Single-Molecule Micromanipulation Studies Of Methylated DNA

Cytosine methylated at the 5-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 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 stiffness and high structural stability of methylated DNA is likely to have significant consequences on the recruitment of proteins recognizing cytosine methylation and DNA packaging. Statement of SignificanceDespite countless structural and functional studies of DNA methylation, a key epigenetic mark in higher organisms, research towards the understanding of DNA intrinsic structural properties in the context of methylation layout representing different epigenetic landscapes is still in its initial stage. We utilize single molecule spectroscopy to analyze the effect of sparse symmetric and asymmetric 5-mC modification on the mechanical stability of long double-stranded DNA. Our findings establish that at physiologically relevant forces and supercoiling densities increased DNA flexibility of non-CpG methylated DNA translates to the high structural stability.