Revealing the Competition between Peeled-Ssdna, Melting Bubbles and S-DNA during DNA Overstretching using Fluorescence Microscopy

Understanding the structural changes occurring in double-stranded (ds)DNA during mechanical strain is essential to build a quantitative picture of how proteins interact and modify DNA. However, the elastic response of dsDNA to tension is only well-understood for forces < 65 pN. Above this force, torsionally unconstrained dsDNA gains ∼70% of its contour length, a process known as overstretching. The structure of overstretched DNA has proved elusive, resulting in a rich and controversial debate in recent years. At the centre of the debate is the question of whether overstretching yields a base-paired elongated structure, known as S-DNA, or instead forms single-stranded (ss)DNA via base-pair cleavage. Here, we show clearly, using a combination of fluorescence microscopy and optical tweezers, that both S-DNA and base-pair melted structures can exist, often concurrently, during overstretching. The balance between the two models is affected strongly by temperature and ionic strength. Moreover, we reveal, for the first time, that base-pair melting can proceed via two entirely different processes: progressive strand unpeeling from a free end in the backbone, or by the formation of ‘bubbles' of ssDNA, nucleating initially in AT-rich regions. We demonstrate that the mechanism of base-pair melting is governed by DNA topology: strand unpeeling is favored when there are free ends in the DNA backbone. Our studies settle a long running debate, and unite the contradictory dogmas of DNA overstretching. These findings have important implications for both medical and biological sciences. Force-induced melting transitions (yielding either peeled-ssDNA or melting bubbles) may play active roles in DNA replication and damage repair. Further, the ability to switch easily from DNA containing melting bubbles to S-DNA may be particularly advantageous in the cell, for instance during the formation of RNA within transcription bubbles. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved

Revealing the Competition between Peeled-Ssdna, Melting Bubbles and S-DNA during DNA Overstretching using Fluorescence Microscopy

Understanding the structural changes occurring in double-stranded (ds)DNA during mechanical strain is essential to build a quantitative picture of how proteins interact and modify DNA. However, the elastic response of dsDNA to tension is only well-understood for forces < 65 pN. Above this force, torsionally unconstrained dsDNA gains ∼70% of its contour length, a process known as overstretching. The structure of overstretched DNA has proved elusive, resulting in a rich and controversial debate in recent years. At the centre of the debate is the question of whether overstretching yields a base-paired elongated structure, known as S-DNA, or instead forms single-stranded (ss)DNA via base-pair cleavage. Here, we show clearly, using a combination of fluorescence microscopy and optical tweezers, that both S-DNA and base-pair melted structures can exist, often concurrently, during overstretching. The balance between the two models is affected strongly by temperature and ionic strength. Moreover, we reveal, for the first time, that base-pair melting can proceed via two entirely different processes: progressive strand unpeeling from a free end in the backbone, or by the formation of ‘bubbles' of ssDNA, nucleating initially in AT-rich regions. We demonstrate that the mechanism of base-pair melting is governed by DNA topology: strand unpeeling is favored when there are free ends in the DNA backbone. Our studies settle a long running debate, and unite the contradictory dogmas of DNA overstretching. These findings have important implications for both medical and biological sciences. Force-induced melting transitions (yielding either peeled-ssDNA or melting bubbles) may play active roles in DNA replication and damage repair. Further, the ability to switch easily from DNA containing melting bubbles to S-DNA may be particularly advantageous in the cell, for instance during the formation of RNA within transcription bubbles. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved

Mechanically probing the folding pathway of single RNA molecules

We study theoretically the denaturation of single RNA molecules by mechanical stretching, focusing on signatures of the (un)folding pathway in molecular fluctuations. Our model describes the interactions between nucleotides by incorporating the experimentally determined free energy rules for RNA secondary structure, while exterior single stranded regions are modeled as freely jointed chains. For exemplary RNA sequences (hairpins and the Tetrahymena thermophila group I intron), we compute the quasi-equilibrium fluctuations in the end-to-end distance as the molecule is unfolded by pulling on opposite ends. Unlike the average quasi-equilibrium force-extension curves, these fluctuations reveal clear signatures from the unfolding of individual structural elements. We find that the resolution of these signatures depends on the spring constant of the force-measuring device, with an optimal value intermediate between very rigid and very soft. We compare and relate our results to recent experiments by Liphardt et al. [Science 292, 733-737 (2001)].Comment: 10 pages, 8 figures, revised version, to be published in Biophys.

DNA as a programmable viscoelastic nanoelement

The two strands of a DNA molecule with a repetitive sequence can pair into many different basepairing patterns. For perfectly periodic sequences, early bulk experiments of Poerschke indicate the existence of a sliding process, permitting the rapid transition between different relative strand positions [Biophys. Chem. 2 (1974) 83]. Here, we use a detailed theoretical model to study the basepairing dynamics of periodic and nearly periodic DNA. As suggested by Poerschke, DNA sliding is mediated by basepairing defects (bulge loops), which can diffuse along the DNA. Moreover, a shear force f on opposite ends of the two strands yields a characteristic dynamic response: An outward average sliding velocity v~1/N is induced in a double strand of length N, provided f is larger than a threshold f_c. Conversely, if the strands are initially misaligned, they realign even against an external force less than f_c. These dynamics effectively result in a viscoelastic behavior of DNA under shear forces, with properties that are programmable through the choice of the DNA sequence. We find that a small number of mutations in periodic sequences does not prevent DNA sliding, but introduces a time delay in the dynamic response. We clarify the mechanism for the time delay and describe it quantitatively within a phenomenological model. Based on our findings, we suggest new dynamical roles for DNA in artificial nanoscale devices. The basepairing dynamics described here is also relevant for the extension of repetitive sequences inside genomic DNA.Comment: 10 pages, 7 figures; final version to appear in Biophysical Journa

The pulsating soft coral Xenia umbellata shows high resistance to warming when nitrate concentrations are low

The resistance of hard corals to warming can be negatively affected by nitrate eutrophication, but related knowledge for soft corals is scarce. We thus investigated the ecophysiological response of the pulsating soft coral Xenia umbellata to different levels of nitrate eutrophication (control = 0.6, medium = 6, high = 37 μM nitrate) in a laboratory experiment, with additional warming (27.7 to 32.8 °C) from days 17 to 37. High nitrate eutrophication enhanced cellular chlorophyll a content of Symbiodiniaceae by 168%, while it reduced gross photosynthesis by 56%. After additional warming, polyp pulsation rate was reduced by 100% in both nitrate eutrophication treatments, and additional polyp loss of 7% d−1 and total fragment mortality of 26% was observed in the high nitrate eutrophication treatment. Warming alone did not affect any of the investigated response parameters. These results suggest that X. umbellata exhibits resistance to warming, which may facilitate ecological dominance over some hard corals as ocean temperatures warm, though a clear negative physiological response occurs when combined with nitrate eutrophication. This study thus confirms the importance of investigating combinations of global and local factors to understand and manage changing coral reefs

The pulsating soft coral Xenia umbellata shows high resistance to warming when nitrate concentrations are low

The resistance of hard corals to warming can be negatively affected by nitrate eutrophication, but related knowledge for soft corals is scarce. We thus investigated the ecophysiological response of the pulsating soft coral Xenia umbellata to different levels of nitrate eutrophication (control = 0.6, medium = 6, high = 37 μM nitrate) in a laboratory experiment, with additional warming (27.7 to 32.8 °C) from days 17 to 37. High nitrate eutrophication enhanced cellular chlorophyll a content of Symbiodiniaceae by 168%, while it reduced gross photosynthesis by 56%. After additional warming, polyp pulsation rate was reduced by 100% in both nitrate eutrophication treatments, and additional polyp loss of 7% d−1 and total fragment mortality of 26% was observed in the high nitrate eutrophication treatment. Warming alone did not affect any of the investigated response parameters. These results suggest that X. umbellata exhibits resistance to warming, which may facilitate ecological dominance over some hard corals as ocean temperatures warm, though a clear negative physiological response occurs when combined with nitrate eutrophication. This study thus confirms the importance of investigating combinations of global and local factors to understand and manage changing coral reefs