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Force regulated dynamics of RPA on a DNA fork

Replication protein A (RPA) is a single-stranded DNA binding protein, involved in most aspects of eukaryotic DNA metabolism. Here, we study the behavior of RPA on a DNA substrate that mimics a replication fork. Using magnetic tweezers we show that both yeast and human RPA can open forked DNA when su...

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Autores principales: Kemmerich, Felix E., Daldrop, Peter, Pinto, Cosimo, Levikova, Maryna, Cejka, Petr, Seidel, Ralf
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4937307/
https://www.ncbi.nlm.nih.gov/pubmed/27016742
http://dx.doi.org/10.1093/nar/gkw187
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author Kemmerich, Felix E.
Daldrop, Peter
Pinto, Cosimo
Levikova, Maryna
Cejka, Petr
Seidel, Ralf
author_facet Kemmerich, Felix E.
Daldrop, Peter
Pinto, Cosimo
Levikova, Maryna
Cejka, Petr
Seidel, Ralf
author_sort Kemmerich, Felix E.
collection PubMed
description Replication protein A (RPA) is a single-stranded DNA binding protein, involved in most aspects of eukaryotic DNA metabolism. Here, we study the behavior of RPA on a DNA substrate that mimics a replication fork. Using magnetic tweezers we show that both yeast and human RPA can open forked DNA when sufficient external tension is applied. In contrast, at low force, RPA becomes rapidly displaced by the rehybridization of the DNA fork. This process appears to be governed by the binding or the release of an RPA microdomain (toehold) of only few base-pairs length. This gives rise to an extremely rapid exchange dynamics of RPA at the fork. Fork rezipping rates reach up to hundreds of base-pairs per second, being orders of magnitude faster than RPA dissociation from ssDNA alone. Additionally, we show that RPA undergoes diffusive motion on ssDNA, such that it can be pushed over long distances by a rezipping fork. Generally the behavior of both human and yeast RPA homologs is very similar. However, in contrast to yeast RPA, the dissociation of human RPA from ssDNA is greatly reduced at low Mg(2+) concentrations, such that human RPA can melt DNA in absence of force.
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spelling pubmed-49373072016-07-11 Force regulated dynamics of RPA on a DNA fork Kemmerich, Felix E. Daldrop, Peter Pinto, Cosimo Levikova, Maryna Cejka, Petr Seidel, Ralf Nucleic Acids Res Nucleic Acid Enzymes Replication protein A (RPA) is a single-stranded DNA binding protein, involved in most aspects of eukaryotic DNA metabolism. Here, we study the behavior of RPA on a DNA substrate that mimics a replication fork. Using magnetic tweezers we show that both yeast and human RPA can open forked DNA when sufficient external tension is applied. In contrast, at low force, RPA becomes rapidly displaced by the rehybridization of the DNA fork. This process appears to be governed by the binding or the release of an RPA microdomain (toehold) of only few base-pairs length. This gives rise to an extremely rapid exchange dynamics of RPA at the fork. Fork rezipping rates reach up to hundreds of base-pairs per second, being orders of magnitude faster than RPA dissociation from ssDNA alone. Additionally, we show that RPA undergoes diffusive motion on ssDNA, such that it can be pushed over long distances by a rezipping fork. Generally the behavior of both human and yeast RPA homologs is very similar. However, in contrast to yeast RPA, the dissociation of human RPA from ssDNA is greatly reduced at low Mg(2+) concentrations, such that human RPA can melt DNA in absence of force. Oxford University Press 2016-07-08 2016-03-25 /pmc/articles/PMC4937307/ /pubmed/27016742 http://dx.doi.org/10.1093/nar/gkw187 Text en © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Nucleic Acid Enzymes
Kemmerich, Felix E.
Daldrop, Peter
Pinto, Cosimo
Levikova, Maryna
Cejka, Petr
Seidel, Ralf
Force regulated dynamics of RPA on a DNA fork
title Force regulated dynamics of RPA on a DNA fork
title_full Force regulated dynamics of RPA on a DNA fork
title_fullStr Force regulated dynamics of RPA on a DNA fork
title_full_unstemmed Force regulated dynamics of RPA on a DNA fork
title_short Force regulated dynamics of RPA on a DNA fork
title_sort force regulated dynamics of rpa on a dna fork
topic Nucleic Acid Enzymes
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4937307/
https://www.ncbi.nlm.nih.gov/pubmed/27016742
http://dx.doi.org/10.1093/nar/gkw187
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