Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching using fluorescence microscopy

Abstract

Mechanical stress plays a key role in many genomic processes, such as DNA replication and transcription. The ability to predict the response of double-stranded (ds) DNA to tension is a cornerstone of understanding DNA mechanics. It is widely appreciated that torsionally relaxed dsDNA exhibits a structural transition at forces of ∼65 pN, known as overstretching, whereby the contour length of the molecule increases by ∼70%. Despite extensive investigation, the structural changes occurring in DNA during overstretching are still generating considerable debate. Three mechanisms have been proposed to account for the increase in DNA contour length during overstretching: strand unpeeling, localized base-pair breaking (yielding melting bubbles), and formation of S-DNA (strand unwinding, while base pairing is maintained). Here we show, using a combination of fluorescence microscopy and optical tweezers, that all three structures can exist, uniting the often contradictory dogmas of DNA overstretching. We visualize and distinguish strand unpeeling and melting-bubble formation using an appropriate combination of fluorescently labeled proteins, whereas remaining B-form DNA is accounted for by using specific fluorescent molecular markers. Regions of S-DNA are associated with domains where fluorescent probes do not bind. We demonstrate that the balance between the three structures of overstretched DNA is governed by both DNA topology and local DNA stability. These findings enhance our knowledge of DNA mechanics and stability, which are of fundamental importance to understanding how proteins modify the physical state of DNA.DNA structure | base-pair stability | DNA melting | single molecules | optical trapping M any cellular processes, such as DNA repair, gene regulation, and genome compaction, induce mechanical stress (1-3). More than a decade ago, single-molecule studies revealed that torsionally unconstrained double-stranded (ds) DNA exhibits a structural transition at tensile forces of ∼65 pN. Here, the DNA molecule gains ∼70% in contour length (L c ), over a narrow force range, a process known as overstretching (4, 5). Such forces are thought to occur in the cell, for example during sister chromatid separation (6). Detailed knowledge of the microscopic change in DNA structure during overstretching is therefore crucial for understanding DNA-protein interactions at the molecular level.

Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

“…Interestingly, a similar effect was reported previously (Candelli et al , 2014) for the assembly process: both nucleation and growth of hRAD51 NPFs are highly tension‐dependent for dsDNA, but independent of tension for ssDNA. This finding can be attributed to the fact that dsDNA is more rigid and resistant to length change (King et al , 2013). The persistence length of ssDNA is more than two orders of magnitude smaller than the one of dsDNA and is much more compliant to protein‐induced structural changes, independent of the tension in the DNA.…”

Coarse-graining DNA for simulations of DNA nanotechnology

“…86 The nature of the overstretched state has been the subject of considerable debate, the two main proposals being force-induced melting by unpeeling 125 and a transition to an overstretched duplex state termed "S-DNA", 126 but with recent evidence suggesting that both mechanisms can occur depending on solution conditions and temperature. 127,128 Interestingly, for our model we only ever see overstretching by unpeeling ( Fig. 6(a)).…”

A polarized view on DNA under tension

“…This range of extensions coincides with the overstretching (OS) transition, in which dsDNA is progressively melted to yield two essentially single-stranded DNA (ssDNA) molecules under low-salt conditions. 35,42,45 What does our observation of constant polarization ratios during this force-induced melting imply? First, it shows that in the OS transition, no significant base pair tilting occurs at or around the intercalated base pairs, which had been suggested as one of the possible structures of (partially) overstretched DNA.…”

“…11 Note that under these conditions, YOYO does not bind to single-stranded DNA (including melting bubbles) or S-DNA, but only to double helical dsDNA. 42,45 Polarized fluorescence Next, the DNA was moved to a buffer channel without YOYO. Here, the concurrent polarized fluorescence and DNA extension experiments were performed by recording fluorescence images with polarization of the excitation light alternating between perpendicular and parallel to the DNA long axis, respectively.…”