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 multiprotein 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.
| Original language | English |
|---|---|
| Pages (from-to) | 2313-2327 |
| Number of pages | 15 |
| Journal | Nucleic Acids Research |
| Volume | 41 |
| Issue number | 4 |
| DOIs | |
| State | Published - Feb 2013 |
Funding
National Institutes of Health operating [R01 GM65484 to W.J.C.; R01 GM46312 to J.A.T.; P01 CA092584 to J.A.T. and W.J.C.]; National Science Foundation [NSF-CAREER MCB-1149521 to I.I.]; Georgia State University (to I.I.); NIH training T32 GM80320 (to C.A.B.); NIH centre grants [P30 ES00267 to the Vanderbilt Center in Molecular Toxicology and P30 CA068485 to the Vanderbilt Ingram Cancer Center]. Computational resources: NSF XSEDE program [CHE110042, in part]; National Energy Research Scientific Computing Center supported by the DOE Office of Science [DE-AC02-05CH11231, in part]. The X-ray scattering technology and applications to the determination of macromolecular shapes and conformations at the SIBYLS beamline (12.3.1) at the Advanced Light Source, Lawrence Berkeley National Laboratory: U.S. Department of Energy (DOE) program Integrated Diffraction Analysis Technologies (IDAT) (in part); NIH grant Macromolecular Insights on Nucleic acids Optimized by Scattering [R01GM105404, in part]. Support from the U.S. Department of Energy for the research at Oak Ridge National Laboratory was provided to the Center for Structural Molecular Biology [FWP ERKP291, Office of Biological and Environmental Research] and the High Flux Isotope Reactor [contract DE-AC05-00OR22725, Scientific User Facilities Division, Office of Basic Energy Sciences]. Funding for open access charge: National Institutes of Health [R01 GM65484].