title: Dynamic binding of replication protein a is required for DNA repair.
abstract: Replication protein A (RPA), the major eukaryotic single-stranded DNA (ssDNA) binding protein, is essential for replication, repair and recombination. High-affinity ssDNA-binding by RPA depends on two DNA binding domains in the large subunit of RPA. Mutation of the evolutionarily conserved aromatic residues in these two domains results in a separation-of-function phenotype: aromatic residue mutants support DNA replication but are defective in DNA repair. We used biochemical and single-molecule analyses, and Brownian Dynamics simulations to determine the molecular basis of this phenotype. Our studies demonstrated that RPA binds to ssDNA in at least two modes characterized by different dissociation kinetics. We also showed that the aromatic residues contribute to the formation of the longer-lived state, are required for stable binding to short ssDNA regions and are needed for RPA melting of partially duplex DNA structures. We conclude that stable binding and/or the melting of secondary DNA structures by RPA is required for DNA repair, including RAD51 mediated DNA strand exchange, but is dispensable for DNA replication. It is likely that the binding modes are in equilibrium and reflect dynamics in the RPA-DNA complex. This suggests that dynamic binding of RPA to DNA is necessary for different cellular functions.
authors.affiliation: Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Ran
authors.initials: R
authors.lastname: Chen
authors.affiliation: Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Shyamal
authors.initials: S
authors.lastname: Subramanyam
authors.affiliation: Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Adrian H
authors.initials: AH
authors.lastname: Elcock
authors.affiliation: Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Spies
authors.affiliation: Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA marc-wold@uiowa.edu.
authors.forename: Marc S
authors.initials: MS
authors.lastname: Wold
mesh_terms: D001665:Binding Sites; D003001:Cloning, Molecular; D053904:DNA Breaks, Single-Stranded; D004260:DNA Repair; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D009154:Mutation; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D051135:Rad51 Recombinase / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D051716:Replication Protein A / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000072760:Single Molecule Imaging; D014741:Video Recording
chemical_list: D004277:DNA, Single-Stranded; C497167:RPA1 protein, human; D011993:Recombinant Fusion Proteins; D051716:Replication Protein A; C495153:RAD51 protein, human; D051135:Rad51 Recombinase