DNA polymerase ¦Á-primase (pol-prim) plays a central role in eukaryotic DNA replication, initiating synthesis on both the leading and lagging strand DNA templates. Pol-prim consists of a primase heterodimer that synthesizes RNA primers, a DNA polymerase that extends them, and a fourth subunit, p68, that is thought to regulate the complex. In the Simian Virus 40 (SV40) replication model, the p68 subunit is required for primosome activity and interacts directly with the viral helicase T antigen (Tag), suggesting a functional link between Tag-p68 interaction and primosome activity. To explore this link, I first mapped the interacting regions of the two proteins and discovered a previously unrecognized N-terminal globular domain of p68 (p68N) that physically interacts with the Tag helicase domain. The solution structure of p68N was determined by NMR spectroscopy in the Chazin lab. The putative Tag-interacting surface on p68N was mapped to a hydrophobic patch surrounded by negative charges. Structure-guided mutagenesis of p68 residues in the interface diminished Tag-p68 interaction and primosome activity of the SV40 replisome. The p68N-docking site on Tag was identified using structure-guided mutagenesis of the Tag helicase surface. A charge reversal substitution in Tag specifically disrupted p68N binding, and hence the SV40 primosome activity. These results collectively suggested that the Tag-p68 interaction is vital for SV40 primosome function. A model is presented for how this interaction regulates primosome activity of the pol-prim complex, providing significant insights into the mechanism of eukaryotic DNA replication initiation.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-11042010-114906 |
| Date | 30 November 2010 |
| Creators | Huang, Hao |
| Contributors | Ellen Fanning, James G. Pattion, Katherine L. Friedman, Brandt F. Eichman, 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-11042010-114906/ |
| 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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