title: Force regulated dynamics of RPA on a DNA fork.
abstract: 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.
authors.affiliation: Institute of Experimental Physics I, Universität Leipzig, Linnéstr. 5, 04103 Leipzig, Germany Institute for Molecular Cell Biology, University of Münster, Schlossplatz 5, D-48149 Münster, Germany.
authors.forename: Felix E
authors.initials: FE
authors.lastname: Kemmerich
authors.affiliation: Institute for Molecular Cell Biology, University of Münster, Schlossplatz 5, D-48149 Münster, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Daldrop
authors.affiliation: Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
authors.forename: Cosimo
authors.initials: C
authors.lastname: Pinto
authors.affiliation: Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
authors.forename: Maryna
authors.initials: M
authors.lastname: Levikova
authors.affiliation: Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
authors.forename: Petr
authors.initials: P
authors.lastname: Cejka
authors.affiliation: Institute of Experimental Physics I, Universität Leipzig, Linnéstr. 5, 04103 Leipzig, Germany Institute for Molecular Cell Biology, University of Münster, Schlossplatz 5, D-48149 Münster, Germany ralf.seidel@physik.uni-leipzig.de.
authors.forename: Ralf
authors.initials: R
authors.lastname: Seidel
mesh_terms: D001696:Biomechanical Phenomena; D003001:Cloning, Molecular; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D055029:Inverted Repeat Sequences; D008274:Magnesium / Q000378:metabolism; D060526:Magnetic Fields; D040542:Mechanotransduction, Cellular; D009691:Nucleic Acid Denaturation; D052898:Optical Tweezers; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D051716:Replication Protein A / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D013500:Surface Tension
chemical_list: D004277:DNA, Single-Stranded; C082728:RFA1 protein, S cerevisiae; C497167:RPA1 protein, human; D011994:Recombinant Proteins; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D008274:Magnesium