title: 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.
authors: LaserLaB Amsterdam and Department of Physics and Astronomy, VU University Amsterdam, 1081 HV Amsterdam, The Netherlands.
authors: Graeme A
authors: GA
authors: King
authors: Peter
authors: P
authors: Gross
authors: Ulrich
authors: U
authors: Bockelmann
authors: Mauro
authors: M
authors: Modesti
authors: Gijs J L
authors: GJ
authors: Wuite
authors: Erwin J G
authors: EJ
authors: Peterman
mesh_terms: D020856:AT Rich Sequence; D020029:Base Pairing; D001667:Binding, Competitive; D055592:Biophysical Phenomena; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D053960:DNA Breaks; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D052898:Optical Tweezers; D009994:Osmolar Concentration; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051716:Replication Protein A / Q000378:metabolism; D013314:Stress, Mechanical; D013816:Thermodynamics
chemical_list: D004277:DNA, Single-Stranded; C497167:RPA1 protein, human; D011993:Recombinant Fusion Proteins; D051716:Replication Protein A; D004247:DNA