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Counterintuitive DNA Sequence Dependence in Supercoiling-Induced DNA Melting

The metabolism of DNA in cells relies on the balance between hybridized double-stranded DNA (dsDNA) and local de-hybridized regions of ssDNA that provide access to binding proteins. Traditional melting experiments, in which short pieces of dsDNA are heated up until the point of melting into ssDNA, h...

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Autores principales: Vlijm, Rifka, v.d. Torre, Jaco, Dekker, Cees
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4625975/
https://www.ncbi.nlm.nih.gov/pubmed/26513573
http://dx.doi.org/10.1371/journal.pone.0141576
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author Vlijm, Rifka
v.d. Torre, Jaco
Dekker, Cees
author_facet Vlijm, Rifka
v.d. Torre, Jaco
Dekker, Cees
author_sort Vlijm, Rifka
collection PubMed
description The metabolism of DNA in cells relies on the balance between hybridized double-stranded DNA (dsDNA) and local de-hybridized regions of ssDNA that provide access to binding proteins. Traditional melting experiments, in which short pieces of dsDNA are heated up until the point of melting into ssDNA, have determined that AT-rich sequences have a lower binding energy than GC-rich sequences. In cells, however, the double-stranded backbone of DNA is destabilized by negative supercoiling, and not by temperature. To investigate what the effect of GC content is on DNA melting induced by negative supercoiling, we studied DNA molecules with a GC content ranging from 38% to 77%, using single-molecule magnetic tweezer measurements in which the length of a single DNA molecule is measured as a function of applied stretching force and supercoiling density. At low force (<0.5pN), supercoiling results into twisting of the dsDNA backbone and loop formation (plectonemes), without inducing any DNA melting. This process was not influenced by the DNA sequence. When negative supercoiling is introduced at increasing force, local melting of DNA is introduced. We measured for the different DNA molecules a characteristic force F (char,) at which negative supercoiling induces local melting of the dsDNA. Surprisingly, GC-rich sequences melt at lower forces than AT-rich sequences: F (char) = 0.56pN for 77% GC but 0.73pN for 38% GC. An explanation for this counterintuitive effect is provided by the realization that supercoiling densities of a few percent only induce melting of a few percent of the base pairs. As a consequence, denaturation bubbles occur in local AT-rich regions and the sequence-dependent effect arises from an increased DNA bending/torsional energy associated with the plectonemes. This new insight indicates that an increased GC-content adjacent to AT-rich DNA regions will enhance local opening of the double-stranded DNA helix.
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spelling pubmed-46259752015-11-06 Counterintuitive DNA Sequence Dependence in Supercoiling-Induced DNA Melting Vlijm, Rifka v.d. Torre, Jaco Dekker, Cees PLoS One Research Article The metabolism of DNA in cells relies on the balance between hybridized double-stranded DNA (dsDNA) and local de-hybridized regions of ssDNA that provide access to binding proteins. Traditional melting experiments, in which short pieces of dsDNA are heated up until the point of melting into ssDNA, have determined that AT-rich sequences have a lower binding energy than GC-rich sequences. In cells, however, the double-stranded backbone of DNA is destabilized by negative supercoiling, and not by temperature. To investigate what the effect of GC content is on DNA melting induced by negative supercoiling, we studied DNA molecules with a GC content ranging from 38% to 77%, using single-molecule magnetic tweezer measurements in which the length of a single DNA molecule is measured as a function of applied stretching force and supercoiling density. At low force (<0.5pN), supercoiling results into twisting of the dsDNA backbone and loop formation (plectonemes), without inducing any DNA melting. This process was not influenced by the DNA sequence. When negative supercoiling is introduced at increasing force, local melting of DNA is introduced. We measured for the different DNA molecules a characteristic force F (char,) at which negative supercoiling induces local melting of the dsDNA. Surprisingly, GC-rich sequences melt at lower forces than AT-rich sequences: F (char) = 0.56pN for 77% GC but 0.73pN for 38% GC. An explanation for this counterintuitive effect is provided by the realization that supercoiling densities of a few percent only induce melting of a few percent of the base pairs. As a consequence, denaturation bubbles occur in local AT-rich regions and the sequence-dependent effect arises from an increased DNA bending/torsional energy associated with the plectonemes. This new insight indicates that an increased GC-content adjacent to AT-rich DNA regions will enhance local opening of the double-stranded DNA helix. Public Library of Science 2015-10-29 /pmc/articles/PMC4625975/ /pubmed/26513573 http://dx.doi.org/10.1371/journal.pone.0141576 Text en © 2015 Vlijm et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Vlijm, Rifka
v.d. Torre, Jaco
Dekker, Cees
Counterintuitive DNA Sequence Dependence in Supercoiling-Induced DNA Melting
title Counterintuitive DNA Sequence Dependence in Supercoiling-Induced DNA Melting
title_full Counterintuitive DNA Sequence Dependence in Supercoiling-Induced DNA Melting
title_fullStr Counterintuitive DNA Sequence Dependence in Supercoiling-Induced DNA Melting
title_full_unstemmed Counterintuitive DNA Sequence Dependence in Supercoiling-Induced DNA Melting
title_short Counterintuitive DNA Sequence Dependence in Supercoiling-Induced DNA Melting
title_sort counterintuitive dna sequence dependence in supercoiling-induced dna melting
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4625975/
https://www.ncbi.nlm.nih.gov/pubmed/26513573
http://dx.doi.org/10.1371/journal.pone.0141576
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