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

King G.A.; Gross P.; Bockelmann U.Modesti M.Wuite G.J.L.Peterman E.J.G.

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

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

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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.

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《Proceedings of the National Academy of Sciences of the United States of America》 |2013年第10期|3859-3864|共6页
作者
King G.A.; Gross P.; Bockelmann U.Modesti M.Wuite G.J.L.Peterman E.J.G.;
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作者单位

LaserLaB Amsterdam and Department of Physics and Astronomy, VU University Amsterdam, 1081 HV Amsterdam, Netherlands;

Laboratoire Nanobiophysique, Unité Mixte de Recherche (UMR) Gulliver 7083, éCole Supérieure de Physique et de Chemie Industrielles de la Ville de Paris, 75005 Paris, France;

Centre de Recherche en Cancérologie de Marseille, CNRS, UMR 7258, F-13009 Marseille, FranceAix-Marseille Université, F-13284 Marseille, France;

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收录信息美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
原文格式 PDF
正文语种英语
中图分类自然科学总论;
关键词
Base-pair stability; DNA melting; DNA structure; Optical trapping; Single molecules;

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

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