W0089: Dynamic Light Scattering Analysis of Full-Length, Human RPA14/32 Dimer: Purification, Crystallization and Self-Association

Replication protein A (RPA) is the eukaryotic single stranded DNA binding protein. Due to the multi dimensional role RPA plays in DNA metabolism, it is of great interest to understand the structure in atomic detail. A NMR solution structure of the N-terminal RPA70 domain (Jacobs et al. (1999) J Biomol NMR 14: 321) and crystal structures at moderate resolution of proteolytic core fragments of RPA70 (Bochkarev et al (1997) Nature 385: 176) and RPA14/32 (Bochkarev et al. (1999) EMBO J 18, 4498) have been reported. Unfortunately, the intact, full-length holoenzyme is very difficult to purify and structural data remains elusive. A soluble dimeric form of RPA is composed of 14 and 32 kDa subunits (RPA14/32). Dynamic light scattering (DLS) analysis was used to improve the purification, stabilization and crystallization of RPA14/32. Increasing concentration of reducing agent in the last stage of purification diminished the size of a secondary peak in the anion exchange chromatograph and promoted a single species in solution. This resulted in decreased polydispersity in the purified protein and enhanced crystallization time. With this homogeneous preparation the reversible association of RPA14/32 into a dimer-of-dimers was demonstrated. Four, different, diffraction-quality, crystal-forms of RPA14/32 were obtained for structure determination. Complete data sets have been collected on hexagonal, spacegroup P6₅ at 2.1Å resolution, and orthorhombic, spacegroup P2₁2₁2₁ at 1.9Å resolution, crystal forms of the protein. Progress on the structure determination of full-length RPA14/32 will be reported.