Force-dependent melting of supercoiled DNA at thermophilic temperatures.
Local DNA opening plays an important role in DNA metabolism as the double-helix must be melted before the information contained within may be accessed. Cells finely tune the torsional state of their genomes to strike a balance between stability and accessibility. For example, while mesophilic life forms maintain negatively superhelical genomes, thermophilic life forms use unique mechanisms to maintain relaxed or even positively supercoiled genomes. Here, we use a single-molecule magnetic tweezers approach to quantify the force-dependent equilibrium between DNA melting and supercoiling at high temperatures populated by Thermophiles. We show that negatively supercoiled DNA denatures at 0.5 pN lower tension at thermophilic vs. mesophilic temperatures. This work demonstrates the ability to monitor DNA supercoiling at high temperature and opens the possibility to perform magnetic tweezers assays on thermophilic systems. The data allow for an estimation of the relative energies of base-pairing and DNA bending as a function of temperature and support speculation as to different general mechanisms of DNA opening in different environments. Lastly, our results imply that average in vivo DNA tensions range between 0.3 and 1.1 pN.
Department of Biochemistry and Molecular Biophysics, Washington University in Saint Louis, 660 South Euclid Avenue, Saint Louis, MO, 63110, USA.
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Galburt
Department of Biochemistry and Molecular Biophysics, Washington University in Saint Louis, 660 South Euclid Avenue, Saint Louis, MO, 63110, USA.
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Tomko
Department of Biochemistry and Molecular Biophysics, Washington University in Saint Louis, 660 South Euclid Avenue, Saint Louis, MO, 63110, USA.
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Stump
Department of Biochemistry and Molecular Biophysics, Washington University in Saint Louis, 660 South Euclid Avenue, Saint Louis, MO, 63110, USA.
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Ruiz Manzano
D004278:DNA, Superhelical / Q000737:chemistry* / Q000378:metabolism; D009691:Nucleic Acid Denaturation; D013696:Temperature
D004278:DNA, Superhelical
DNA melting; DNA superhelicity; DNA tension; Magnetic tweezers; Single-molecule