The replication of DNA occurs through a complex series of steps involving the coordinated action of many proteins. DNA polymerase alpha/primase (pol-prim) is a critical DNA replication factor that synthesizes short RNA-DNA primers on the leading and lagging strands of DNA being actively replicated. Pol-prim contains a DNA polymerase subunit (p180), an accessory subunit (p68), and two DNA primase subunits (p58 and p48). This dissertation describes the biochemical and structural characterization of two folded, globular pol-prim domains: p58C and p68N. Sub-cloning, bacterial expression, purification, and analysis of p58C revealed an essential iron-sulfur cluster, the first such cofactor identified in a DNA replication protein. Sub-cloning, bacterial expression, and NMR analysis enabled the determination of the solution structure of p68N. The binding of p68N to the SV40 large T antigen helicase domain was measured by isothermal titration calorimetry and the structure was used to model and test the molecular basis for this interaction. These results enabled an updated model to be generated for the action of pol-prim in SV40 DNA replication.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-07222008-151325 |
| Date | 28 July 2008 |
| Creators | Weiner, Brian Edward |
| Contributors | Ellen Fanning, Brandt F. Eichman, David Cortez, Richard N. Armstrong, Walter J. Chazin |
| 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-07222008-151325/ |
| 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. |
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