title: Structure of the RPA trimerization core and its role in the multistep DNA-binding mechanism of RPA.
abstract: The human single-stranded DNA-binding protein, replication protein A (RPA) binds DNA in at least two different modes: initial [8-10 nucleotides (nt)] and stable ( approximately 30 nt). Switching from 8 to 30 nt mode is associated with a large conformational change. Here we report the 2.8 A structure of the RPA trimerization core comprising the C-terminal DNA-binding domain of subunit RPA70 (DBD-C), the central DNA-binding domain of subunit RPA32 (DBD-D) and the entire RPA14 subunit. All three domains are built around a central oligonucleotide/oligosaccharide binding (OB)-fold and flanked by a helix at the C-terminus. Trimerization is mediated by three C-terminal helices arranged in parallel. The OB-fold of DBD-C possesses unique structural features; embedded zinc ribbon and helix-turn-helix motifs. Using time-resolved proteolysis with trypsin, we demonstrate that the trimerization core does not contribute to the binding with substrates of 10 nt, but interacts with oligonucleotides of 24 nt. Taken together, our data indicate that switching from 8-10 to 30 nt mode is mediated by DNA binding with the trimerization core.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA.
authors.forename: Elena
authors.initials: E
authors.lastname: Bochkareva
authors.forename: Sergey
authors.initials: S
authors.lastname: Korolev
authors.forename: Susan P
authors.initials: SP
authors.lastname: Lees-Miller
authors.forename: Alexey
authors.initials: A
authors.lastname: Bochkarev
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D051716:Replication Protein A
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D051716:Replication Protein A