title: Counterintuitive DNA Sequence Dependence in Supercoiling-Induced DNA Melting.
abstract: The metabolism of DNA in cells relies on the balance between hybridized double-stranded DNA (dsDNA) and local de-hybridized regions of ssDNA that provide access to binding proteins. Traditional melting experiments, in which short pieces of dsDNA are heated up until the point of melting into ssDNA, have determined that AT-rich sequences have a lower binding energy than GC-rich sequences. In cells, however, the double-stranded backbone of DNA is destabilized by negative supercoiling, and not by temperature. To investigate what the effect of GC content is on DNA melting induced by negative supercoiling, we studied DNA molecules with a GC content ranging from 38% to 77%, using single-molecule magnetic tweezer measurements in which the length of a single DNA molecule is measured as a function of applied stretching force and supercoiling density. At low force (<0.5pN), supercoiling results into twisting of the dsDNA backbone and loop formation (plectonemes), without inducing any DNA melting. This process was not influenced by the DNA sequence. When negative supercoiling is introduced at increasing force, local melting of DNA is introduced. We measured for the different DNA molecules a characteristic force Fchar, at which negative supercoiling induces local melting of the dsDNA. Surprisingly, GC-rich sequences melt at lower forces than AT-rich sequences: Fchar = 0.56pN for 77% GC but 0.73pN for 38% GC. An explanation for this counterintuitive effect is provided by the realization that supercoiling densities of a few percent only induce melting of a few percent of the base pairs. As a consequence, denaturation bubbles occur in local AT-rich regions and the sequence-dependent effect arises from an increased DNA bending/torsional energy associated with the plectonemes. This new insight indicates that an increased GC-content adjacent to AT-rich DNA regions will enhance local opening of the double-stranded DNA helix.
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Delft, the Netherlands.
authors.forename: Rifka
authors.initials: R
authors.lastname: Vlijm
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Delft, the Netherlands.
authors.forename: Jaco
authors.initials: J
authors.lastname: V D Torre
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Delft, the Netherlands.
authors.forename: Cees
authors.initials: C
authors.lastname: Dekker
mesh_terms: D001482:Base Composition; D004278:DNA, Superhelical / Q000737:chemistry*; D020862:GC Rich Sequence; D060526:Magnetic Fields; D009691:Nucleic Acid Denaturation
chemical_list: D004278:DNA, Superhelical