Sample Preparation Methods to Analyze DNA-Induced Structural Changes in Replication Protein A

Abstract

Propagation and maintenance of the cellular genome are among the most fundamental cellular processes, encompassing pathways associated with DNA replication, damage response, and repair. Replication Protein A (RPA), the primary single-stranded DNA-binding protein (SSB) in eukaryotes, serves to protect ssDNA generated during these events and to recruit and organize other DNA-processing factors requiring access to ssDNA substrates. RPA engages ssDNA in distinct, progressive binding modes, which are thought to correspond to different functional states of the protein during the course of DNA processing. Structural characterization of these unique complexes has remained challenging, however, as RPA is a multi-domain protein characterized by a flexible, modular organization. Biophysical approaches that are well suited to probing time-varying architectures, such as NMR and small-angle X-ray and neutron scattering (SAXS/SANS), when integrated with computational methods, can provide critical insights into the architectural changes associated with RPA's different DNA-binding modes. The success of these methods, however, is highly contingent upon the purity, homogeneity, and stability of the sample under study. Here we describe a basic protocol for characterizing and optimizing sample conditions for RPA/ssDNA complexes prior to study by SAXS and/or SANS.