Effect of Methylation on the Nanomechanical Properties of Double-Stranded DNA

Abstract

In mammalian cells 60-90% of cytosines in the genome are methylated. The methylation sites are unevenly distributed and are often found in clusters called “CpG islands”. Approximately 70% of promoters in the human genome contain or are preceeded by CG-rich regions, suggesting that methylation may be important in gene regulation. Cyclization-kinetic and nucleosome-binding assays suggest that methylation may significantly affect DNA flexibility. However, a direct effect of methylation on the mechanics of DNA is yet unknown. To investigate the impact of methylation on DNA mechanics, here we manipulated single molecules of methylated dsDNA and compared their nanomechanical properties with those of unmethylated DNA.A 3500-base-pair sequence of lambda-phage DNA composed almost entirely of CpG islands was cloned by using PCR containing dm5CTP to produce the fully methylated product. Individual DNA molecules were mechanically manipulated in stretch and relaxation cycles by using custom-built dual-beam counter-propagating optical tweezers. Force versus extension data were fitted with the extensible wormlike-chain model to obtain the contour length, the persistence length (entropic component of rigidity) and the stretch modulus (enthalpic component of rigidity) of dsDNA. Methylation reduced the contour length and stretch modulus of dsDNA from 1036±22 nm to 966±8 nm and from 1225±115 pN to 373±30 pN, respectively. Persistence length was 34±2 nm for the non-methylated and 35±2 nm in case of the hypermethylated DNA. The observed changes may be caused by a complex shift in tertiary structure, accounting for both the reduction of the contour length and the increase of the intrinsic compliance of the dsDNA chain. The methylation-induced effects on the nanomechanical properties of dsDNA may play an important role in the regulation of steric access to its sequence-specific sites.