title: Diffusion of human replication protein A along single-stranded DNA.
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 D1~5000nt(2) 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.
authors: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110, USA.
authors: Binh
authors: B
authors: Nguyen
authors: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110, USA.
authors: Joshua
authors: J
authors: Sokoloski
authors: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110, USA.
authors: Roberto
authors: R
authors: Galletto
authors: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110, USA.
authors: Elliot L
authors: EL
authors: Elson
authors: Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
authors: Marc S
authors: MS
authors: Wold
authors: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110, USA. Electronic address: Lohman@wustl.edu.
authors: Timothy M
authors: TM
authors: Lohman
mesh_terms: D002232:Carbocyanines / Q000737:chemistry; D004260:DNA Repair / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics* / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry; D015321:Gene Rearrangement / Q000235:genetics; D006801:Humans; D009691:Nucleic Acid Denaturation / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011995:Recombination, Genetic; D051716:Replication Protein A / Q000737:chemistry* / Q000648:ultrastructure*
chemical_list: D002232:Carbocyanines; D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D051716:Replication Protein A; C089187:cyanine dye 3
keywords: DNA hairpin melting; RPA; diffusion coefficient; dynamics; single-molecule fluorescence