title: Preparation of the modular multi-domain protein RPA for study by NMR spectroscopy.
abstract: The integrity and propagation of the genome depend upon the fidelity of DNA processing events, such as replication, damage recognition, and repair. Requisite to the numerous biochemical tasks required for DNA processing is the generation and manipulation of single-stranded DNA (ssDNA). As the primary eukaryotic ssDNA-binding protein, Replication Protein A (RPA) protects ssDNA templates from stray nuclease cleavage and untimely reannealment. More importantly, RPA also serves as a platform for organizing access to ssDNA for readout of the genetic code, recognition of aberrations in DNA, and processing by enzymes. We have proposed that RPA's ability to adapt to such a broad spectrum of multiprotein machinery arises in part from its modular organization and interdomain flexibility. While requisite for function, RPA's modular flexibility has presented many challenges to providing a detailed characterization of the dynamic architecture of the full-length protein. To enable the study of RPA's interdomain dynamics and responses to ssDNA binding by biophysical methods including NMR spectroscopy, we have successfully produced recombinant full-length RPA in milligram quantities at natural abundance and enriched with NMR-active isotopes.
authors.affiliation: Departments of Biochemistry and Chemistry, Center for Structural Biology, Vanderbilt University, Nashville, TN, USA.
authors.forename: Chris A
authors.initials: CA
authors.lastname: Brosey
authors.forename: Marie-Eve
authors.initials: ME
authors.lastname: Chagot
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Chazin
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods; D002472:Cell Transformation, Viral; D002850:Chromatography, Gel; D003470:Culture Media / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051716:Replication Protein A / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D003470:Culture Media; D004277:DNA, Single-Stranded; D051716:Replication Protein A