Structural Dynamics and Single-Stranded DNA Binding Activity of the Three N-Terminal Domains of the Large Subunit of Replication Protein A from Small Angle X-ray Scattering

Abstract

Replication Protein A (RPA) is the primary eukaryotic ssDNA binding protein utilized in diverse DNA transactions in the cell. RPA is a heterotrimeric protein with seven globular domains connected by flexible linkers, which enable substantial inter-domain motion that is essential to its function. Small angle X-ray scattering (SAXS) experiments on two multi-domain constructs from the Nterminus of the large subunit (RPA70) were used to examine the structural dynamics of these domains and their response to the binding of ssDNA. The SAXS data combined with molecular dynamics simulations reveal substantial interdomain flexibility for both RPA70AB (the tandem high affinity ssDNA binding domains A and B connected by a 10-residue linker) and RPA70NAB (RPA70AB extended by a 70-residue linker to the RPA70N protein interaction domain). Binding of ssDNA to RPA70NAB reduces the interdomain flexibility between the A and B domains, but has no effect on RPA70N. These studies provide the first direct measurements of changes in orientation of these three RPA domains upon binding ssDNA. The results support a model in which RPA70N remains structurally independent of RPA70AB in the DNA bound state and therefore freely available to serve as a protein recruitment module.RPA is the primary eukaryotic ssDNA binding protein utilized for diverse DNA transactions in the replication and maintenance of the genome (reviewed by Fanning and coworkers [1]). RPA functions by binding and protecting ssDNA from degradation by endonucleases, inhibiting formation of ssDNA secondary structure, and providing a scaffold for DNA processing machinery by interacting with numerous DNA processing proteins. RPA biochemical functions and biological activities have been intensively investigated and the structures of its domains determined [2][3][4][5][6][7][8][9]. Despite this detailed information, the mechanisms for RPA function remain poorly understood, largely due to the inherent difficulties of *To whom correspondence should be addressed: Center for Structural Biology, Vanderbilt University, 465 21 st Avenue, Suite 5140, Nashville, Telephone: (615) 936-2210; Fax: (615) 936-2211; walter.chazin@vanderbilt.edu.• These authors contributed equally to this work.Experimental and theoretical scattering profiles, P(r) functions, SAXS envelopes and atomic models will be deposited in the BIOISIS database (www.bioisis.net) under accession code 61. SUPPORTING INFORMATION AVAILABLE. SEC profiles and Guinier analysis for RPA70AB, RPA70NAB and their ssDNA complexes. This material is available free of charge via the internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 April 6. Published in final edited form as:Biochemistry. 2010 April 6; 49(13): 2880-2889. doi:10.1021/bi9019934. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript characterizing proteins with modular organization and the fact that RPA function is integrated within complex multi-protein machinery.RPA is a mod...

Structural organization of the functional domains of Clostridium difficile toxins A and B

“…We interpret this to mean that the TcdB receptorbinding domain is able to adopt multiple orientations with respect to the rest of the molecule. This heterogeneity is the likely explanation for why the molecular envelope for TcdB obtained by SAXS differs from what we observe (17) because ab initio envelope calculations from scattering data can be problematic in flexible systems in which domain orientations differ between conformers (25,26).…”

BID Binds to Replication Protein A and Stimulates ATR Function following Replicative Stress

“…Motion between domains allows for optimal association with DNA and associated proteins during DNA processing (2,8,24,42,48). RPA binds ssDNA using four OB-fold domains that bind sequentially to DNA with a 5Ј-3Ј polarity (20,28).…”

The Structure and Function of Replication Protein A in DNA Replication

“…For parts a , b and c , PDB IDs 1QUQ, 2PI2 and 2PQA were used, respectively. For parts d and e , PDB ID 1LIO was used Small angle x-ray scattering (SAXS) has been used to study the structural dynamics of the N-terminal half of RPA1 (including domains RPA1-F, -A and -B) when bound to ssDNA (Pretto et al 2010 ) . Consistent with previous reports, SAXS data indicate that binding of ssDNA to RPA1-FAB reduces the interdomain fl exibility between RPA1-A and -B but has no effect on RPA1-F which is available for protein interactions.…”