Overstretching DNA does not require peeling

DNA’s extension increases by 70%

Double-stranded DNA with no nicks or free ends is attached to a glass slide and a tiny bead, then gently pulled by an optical trap until the DNA “overstretches” and becomes 70% longer. The Perkins group has shown that peeling from nicks or free ends is not the only possible mechanism at play in DNA overstretching at 65 pN.  Figure credit: Brad Baxley, JILA.

DNA’s extension increases by 70% in a narrow force window at 65 pN, a phenomena called 'over stretching'. The state of the DNA after overstretching has been controversial since the discovery of overstretching in 1996. One group of scientists advocate for S-DNA, where the rise per base pair and the tilt is significantly altered. Another group of scientists argue that this is completely wrong, and that overstretching DNA is really a force-induced melting.

Given this ongoing uncertainty, we sought to directly test if the generation of single stranded (ss) DNA via a peeling mechanism – the leading mechanism for overstretching DNA (PNAS, 2009, 106, 18231) – is necessary for overstretching DNA. However, to date, limitations in substrates have led to a discontinuous transition between stretching DNA that has no free ends (Fo = 110 pN) and DNA with nicks or free ends (Fo = 65 pN) because assays containing no free ends were both topologically closed and torsionally constrained. We developed a new topologically closed but rotationally unconstrained assay by binding DNA at one end to a surface by both strands and the other end to a bead via an internal biotin embedded in a 5-nucleotide loop.

In summary, DNA in the topologically closed assay overstretched at the canonical 65 pN, but lackedhysteresis.  Hence, ssDNA generation from nicks or free ends – the leading mechanism for overstretching DNA is not a compulsory step in overstretching. Rather, such peeling is the primary cause of hysteresis in the force-extension curve. We hope that these new findings on the mechanism of overstretching DNA will narrow the range of the ongoing debate about overstretched DNA.

Related Paper

JACS coverOverstretching DNA at 65 pN Does Not Require Peeling from Free Ends or Nicks, D. Hern Paik and Thomas T. Perkins