Diffusion of Human Replication Protein A along Single-Stranded DNA

Binh Nguyen; Joshua Sokoloski; Roberto Galletto; Elliot L Elson; Marc S Wold; Timothy M Lohman

doi:10.1016/j.jmb.2014.07.014

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Diffusion of Human Replication Protein A along Single-Stranded DNA

Journal article

Peer reviewed

Diffusion of Human Replication Protein A along Single-Stranded DNA

Binh Nguyen, Joshua Sokoloski, Roberto Galletto, Elliot L Elson, Marc S Wold and Timothy M Lohman

Journal of molecular biology, Vol.426(19), pp.3246-3261

09/23/2014

DOI: 10.1016/j.jmb.2014.07.014

PMCID: PMC4150847

PMID: 25058683

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http://doi.org/10.1016/j.jmb.2014.07.014View

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Abstract

Replication protein A (RPA) is a eukaryotic single-stranded DNA (ssDNA) binding protein that plays critical roles in most aspects of genome maintenance, including replication, recombination and repair. RPA binds ssDNA with high affinity, destabilizes DNA secondary structure and facilitates binding of other proteins to ssDNA. However, RPA must be removed from or redistributed along ssDNA during these processes. To probe the dynamics of RPA–DNA interactions, we combined ensemble and single-molecule fluorescence approaches to examine human RPA (hRPA) diffusion along ssDNA and find that an hRPA heterotrimer can diffuse rapidly along ssDNA. Diffusion of hRPA is functional in that it provides the mechanism by which hRPA can transiently disrupt DNA hairpins by diffusing in from ssDNA regions adjacent to the DNA hairpin. hRPA diffusion was also monitored by the fluctuations in fluorescence intensity of a Cy3 fluorophore attached to the end of ssDNA. Using a novel method to calibrate the Cy3 fluorescence intensity as a function of hRPA position on the ssDNA, we estimate a one-dimensional diffusion coefficient of hRPA on ssDNA of D1~5000nt2 s−1 at 37°C. Diffusion of hRPA while bound to ssDNA enables it to be readily repositioned to allow other proteins access to ssDNA. [Display omitted] •RPA binds ssDNA tightly and is essential for genome maintenance.•hRPA can diffuse along ssDNA.•hRPA can transiently melt DNA hairpins by diffusing in from adjacent ssDNA.•Hairpin melting is more efficient if RPA diffuses in from the 5′ side of a hairpin.•We estimate an hRPA diffusion coefficient (5000nt2 s−1 at 37°C) using a novel method.

RPA

dynamics

single-molecule fluorescence

DNA hairpin melting

diffusion coefficient

Details

Title: Subtitle: Diffusion of Human Replication Protein A along Single-Stranded DNA
Creators: Binh Nguyen - Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110, USA
Joshua Sokoloski - Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110, USA
Roberto Galletto - Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110, USA
Elliot L Elson - Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110, USA
Marc S Wold - Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA
Timothy M Lohman - Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110, USA
Resource Type: Journal article
Publication Details: Journal of molecular biology, Vol.426(19), pp.3246-3261
DOI: 10.1016/j.jmb.2014.07.014
PMID: 25058683
PMCID: PMC4150847
NLM abbreviation: J Mol Biol
ISSN: 0022-2836
eISSN: 1089-8638
Publisher: Elsevier Ltd
Grant note: DOI: 10.13039/100000002, name: National Institutes of Health, award: GM030498, GM044721, GM098509, HL109505
Language: English
Date published: 09/23/2014
Academic Unit: Radiation Oncology; Biochemistry and Molecular Biology
Record Identifier: 9984025296002771

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