title: A small molecule directly inhibits the p53 transactivation domain from binding to replication protein A.
abstract: Replication protein A (RPA), essential for DNA replication, repair and DNA damage signalling, possesses six ssDNA-binding domains (DBDs), including DBD-F on the N-terminus of the largest subunit, RPA70. This domain functions as a binding site for p53 and other DNA damage and repair proteins that contain amphipathic alpha helical domains. Here, we demonstrate direct binding of both ssDNA and the transactivation domain 2 of p53 (p53TAD2) to DBD-F, as well as DBD-F-directed dsDNA strand separation by RPA, all of which are inhibited by fumaropimaric acid (FPA). FPA binds directly to RPA, resulting in a conformational shift as determined through quenching of intrinsic tryptophan fluorescence in full length RPA. Structural analogues of FPA provide insight on chemical properties that are required for inhibition. Finally, we confirm the inability of RPA possessing R41E and R43E mutations to bind to p53, destabilize dsDNA and quench tryptophan fluorescence by FPA, suggesting that protein binding, DNA modulation and inhibitor binding all occur within the same site on DBD-F. The disruption of p53-RPA interactions by FPA may disturb the regulatory functions of p53 and RPA, thereby inhibiting cellular pathways that control the cell cycle and maintain the integrity of the human genome.
authors.affiliation: Department of Oral Biology, College of Dentistry, University of Nebraska Medical Center, Lincoln, NE 68583, USA.
authors.forename: Jason G
authors.initials: JG
authors.lastname: Glanzer
authors.forename: Katie A
authors.initials: KA
authors.lastname: Carnes
authors.forename: Patricia
authors.initials: P
authors.lastname: Soto
authors.forename: Shengqin
authors.initials: S
authors.lastname: Liu
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Parkhurst
authors.forename: Gregory G
authors.initials: GG
authors.lastname: Oakley
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D001667:Binding, Competitive; D004277:DNA, Single-Stranded / Q000378:metabolism; D004224:Diterpenes / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D051716:Replication Protein A / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004277:DNA, Single-Stranded; D004224:Diterpenes; C497167:RPA1 protein, human; D051716:Replication Protein A; D016159:Tumor Suppressor Protein p53; C001265:fumaropimaric acid