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.
<p>
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.
<p>
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.
<p>
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.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-10282011-155051 |
| Date | 31 October 2011 |
| Creators | Brosey, Chris Arlen |
| Contributors | David Cortez, Walter J. Chazin, Charles Sanders, Jens Meiler, Ellen Fanning, Brandt Eichman |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-10282011-155051/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
Page generated in 0.0015 seconds