title: A new structural framework for integrating replication protein A into DNA processing machinery.
abstract: By coupling the protection and organization of single-stranded DNA (ssDNA) with recruitment and alignment of DNA processing factors, replication protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA coordinates biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA's DNA-binding activity, combining small-angle X-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA's DNA-binding core. The scattering data reveal compaction promoted by DNA binding; DNA-free RPA exists in an ensemble of states with inter-domain mobility and becomes progressively more condensed and less dynamic on binding ssDNA. Our results contrast with previous models proposing RPA initially binds ssDNA in a condensed state and becomes more extended as it fully engages the substrate. Moreover, the consensus view that RPA engages ssDNA in initial, intermediate and final stages conflicts with our data revealing that RPA undergoes two (not three) transitions as it binds ssDNA with no evidence for a discrete intermediate state. These results form a framework for understanding how RPA integrates the ssDNA substrate into DNA processing machinery, provides substrate access to its binding partners and promotes the progression and selection of DNA processing pathways.
authors.affiliation: Department of Biochemistry, Center for Structural Biology, Vanderbilt University, Nashville, TN 37232, USA.
authors.forename: Chris A
authors.initials: CA
authors.lastname: Brosey
authors.forename: Chunli
authors.initials: C
authors.lastname: Yan
authors.forename: Susan E
authors.initials: SE
authors.lastname: Tsutakawa
authors.forename: William T
authors.initials: WT
authors.lastname: Heller
authors.forename: Robert P
authors.initials: RP
authors.lastname: Rambo
authors.forename: John A
authors.initials: JA
authors.lastname: Tainer
authors.forename: Ivaylo
authors.initials: I
authors.lastname: Ivanov
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Chazin
mesh_terms: D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D033363:Neutron Diffraction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051716:Replication Protein A / Q000737:chemistry* / Q000378:metabolism; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
chemical_list: D004277:DNA, Single-Stranded; D051716:Replication Protein A