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 DNA to tension is a cornerstone of our understanding of DNA mechanics. It is widely appreciated that torsionally relaxed DNA exhibits a transition at forces of ~65 pN, whereby it gains ~70% of its contour length. However, there is still considerable debate over the structural change in DNA during such ‘overstretching’. Three mechanisms have been proposed to account for the increase in DNA contour length during overstretching: strand unpeeling, localized base-pair breaking (melting bubbles) and S-DNA (strand unwinding, while retaining base-pairing). Here we show, using a combination of fluorescence microscopy and optical tweezers, that all three phases can exist, uniting the often contradictory dogmas of DNA overstretching. We reveal that these three structures can each be identified by their unique signatures in force extension measurements. Moreover, we visualize and distinguish strand unpeeling and melting bubbles using an appropriate combination of fluorescently labeled proteins, while remaining B-form DNA is accounted for using specific fluorescent molecular markers. Regions of S-DNA are associated with domains where fluorescent probes do not bind. By considering salt concentration, base-pair sequence and the topology of the DNA backbone, we demonstrate that the balance between the three phases of overstretched DNA is governed by the interplay between electrostatic strand repulsion and base-pairing interactions. The findings are of importance to the study of DNA-protein interactions, and guide theoretical models for predicting DNA structure and conformation