Investigating the architectural basis of RPA quaternary remodeling upon binding ssDNA

Abstract

The integrity and propagation of the genome depends 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 serves as a platform for organizing access to ssDNA for readout of the genetic code, recognition of aberrations in DNA, and processing by enzymes.

As a universal participant in DNA processing, RPA must interact with a wide array of structurally unique multi-protein complexes and ssDNA substrates. The flexible, modular organization of the protein is thought to be critical for enabling such structural adaptability. Despite the availability of high-resolution x-ray and NMR structures of individual RPA domains, the dynamic interdomain organization of full-length RPA and the accompanying structural alterations imposed by DNA processing have not been extensively characterized.

The scope of this dissertation research has focused upon probing the solution arrangement of modular domains within full-length RPA and the rearrangement of this inter-domain architecture as RPA engages ssDNA substrates. NMR studies on the full-length protein resolve conflicting views of RPA architecture within the literature, indicating an absence of inter-domain contacts and favoring a model for flexible independence of RPA domains. NMR 15N relaxation studies on select tandem domain fragments from RPA provide a detailed biophysical description of the inter-domain dynamics indicated by NMR studies on the full-length protein, revealing that the rotational motion of modular domains is largely dependent upon the length of the interconnecting linker, as well as the presence of ssDNA substrate. Results from small-angle x-ray scattering (SAXS) studies on RPA's DNA-binding core provide insight into the inter-domain rearrangements that accompany RPA as it proceeds through its three modes of DNA-binding, suggesting that the DNA-binding core progressively compacts as it proceeds through initial and intermediate binding modes, but loses this compaction upon transitioning to the final binding mode.

This work has provided the first view of the global disposition of RPA's inter-domain organization and how RPA's dynamic quaternary structure is refashioned upon binding ssDNA. These findings serve as an essential prerequisite to understanding how RPA coordinates access to ssDNA templates and regulates progression of DNA processing events.