Human replication protein A (RPA) is a single-stranded DNA (ssDNA) binding protein involved in DNA metabolism. RPA binds ssDNA transiently during initiation of DNA replication. When this dissertation research began, the mechanisms of RPA loading and displacement were not known. Two SV40 T antigen-binding sites on RPA, DNA binding domains A and B of RPA70 (RPA70AB) and C-terminus of RPA32 (RPA32C) have been defined. The origin DNA-binding domain (OBD) of T antigen binds to both sites. Physical interaction between T antigen OBD and RPA70AB was required for the loading of RPA onto ssDNA during initiation of SV40 DNA replication. T antigen formed a ternary complex with RPA and 8-mer ssDNA, but was released from RPA-ssDNA complex when longer ssDNA was available. Thus the ternary complex is a key intermediate for RPA loading. Although RPA32C is not involved in this process, it is crucial for RPA displacement from ssDNA by primosome activity. A charge reversal mutant of RPA32C showed reduced binding affinity for T antigen OBD. The same mutation introduced into intact RPA impaired initiation of replication and primosome activity. Based on these results, a dynamic model of RPA function in the initiation of SV40 DNA replication is proposed, in which successive protein-mediated remodeling of RPA facilitates its binding to ssDNA during origin DNA unwinding and its dissociation from ssDNA upon completion of primer synthesis.
Topoisomerase IIβ binding protein 1 (TopBP1), which stimulates SV40 DNA replication, was found to physically interact with T antigen and DNA polymerase α-primase (pol-prim).
In this dissertation, Chapter I presents an introduction to SV40 DNA replication as a model system. Chapters II and III are present two publications in which my major research results are included. Chapter IV is a summary and discussion of the functional dynamics of RPA in viral DNA replication. The appendix includes unpublished evidence of TopBP1 interaction with T antigen and pol-prim, which may participate in SV40 DNA replication in infected cells.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-03252008-092729 |
| Date | 04 April 2008 |
| Creators | Jiang, Xiaohua |
| Contributors | James G. Patton, Walter Chazin, Mark Denison, Katherine Friedman, Ellen Fanning |
| 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-03252008-092729/ |
| 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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