title: Structural mechanism of RPA loading on DNA during activation of a simple pre-replication complex.
abstract: We report that during activation of the simian virus 40 (SV40) pre-replication complex, SV40 T antigen (Tag) helicase actively loads replication protein A (RPA) on emerging single-stranded DNA (ssDNA). This novel loading process requires physical interaction of Tag origin DNA-binding domain (OBD) with the RPA high-affinity ssDNA-binding domains (RPA70AB). Heteronuclear NMR chemical shift mapping revealed that Tag-OBD binds to RPA70AB at a site distal from the ssDNA-binding sites and that RPA70AB, Tag-OBD, and an 8-nucleotide ssDNA form a stable ternary complex. Intact RPA and Tag also interact stably in the presence of an 8-mer, but Tag dissociates from the complex when RPA binds to longer oligonucleotides. Together, our results imply that an allosteric change in RPA quaternary structure completes the loading reaction. A mechanistic model is proposed in which the ternary complex is a key intermediate that directly couples origin DNA unwinding to RPA loading on emerging ssDNA.
authors.affiliation: Department of Biological Sciences, Vanderbilt University, Nashville, TN 37232-8725, USA.
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Jiang
authors.forename: Vitaly
authors.initials: V
authors.lastname: Klimovich
authors.forename: Alphonse I
authors.initials: AI
authors.lastname: Arunkumar
authors.forename: Erik B
authors.initials: EB
authors.lastname: Hysinger
authors.forename: Yingda
authors.initials: Y
authors.lastname: Wang
authors.forename: Robert D
authors.initials: RD
authors.lastname: Ott
authors.forename: Gulfem D
authors.initials: GD
authors.lastname: Guler
authors.forename: Brian
authors.initials: B
authors.lastname: Weiner
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Chazin
authors.forename: Ellen
authors.initials: E
authors.lastname: Fanning
mesh_terms: D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry*; D001665:Binding Sites; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D025941:Protein Interaction Mapping; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D018741:Replication Origin; D051716:Replication Protein A / Q000737:chemistry*; D055672:Static Electricity
chemical_list: D000952:Antigens, Polyomavirus Transforming; D004277:DNA, Single-Stranded; C497167:RPA1 protein, human; D051716:Replication Protein A