Mcm10 is an essential eukaryotic DNA replication protein required for assembly and progression of the replication fork. Specifically, Mcm10 is required for the association of several replication proteins, including DNA polymerase α (pol α), with chromatin. However, the significance of these interactions, and the specific role of Mcm10 during replication initiation is unclear. To address this gap in knowledge, we have begun a structure/function analysis of Xenopus laevis Mcm10 (xMcm10). Chapter 2 describes Mcm10s domain structure which is composed of three structural and functional regions. The amino-terminal domain (NTD) forms a dimerization motif, while the internal (ID) and carboxy-terminal (CTD) domains of Mcm10 physically interact with both single-stranded (ss)DNA and the catalytic p180 subunit of pol α. Chapter 3 describes the crystal structure determination of xMcm10-ID as well as NMR spectroscopy to map the binary interfaces between xMcm10-ID and ssDNA. In chapter 4, the mechanism by which Mcm10-ID interacts with pol α is investigated using X-ray crystallography, NMR spectroscopy, isothermal titration calorimetry, and fluorescence anisotropy. In addition, the manner in which the ID and CTD operate together to interact with DNA and pol α are investigated. Collectively, the results presented here provide the first mechanistic insight into how Mcm10 might use a hand-off mechanism to load and stabilize pol α within the replication fork. We propose that the modularity of the protein architecture, with discrete domains for dimerization and for binding to DNA and pol α, provides an effective means for coordinating the biochemical activities of Mcm10 within the replisome.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-07062009-173137 |
| Date | 14 July 2009 |
| Creators | Warren Jr., Eric Mason |
| Contributors | Walter Chazin, Ellen Fanning, Daniel Kaplan, Brandt Eichman, Al Beth |
| 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-07062009-173137/ |
| 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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