Human papillomavirus type 16 (HPVI6) is a causative agent of cervical cancer. The HPV16 viral transcription/replication factor E2 is essential for the viral life cycle. The function of E2 is dependenton the interaction with cellular partner proteins. The amino- terminal domain of E2 is an acidic protein-protein interaction domain essential for all of the functions of E2. A yeast-2-hybrid screen using the amino-terminal domain of HPV16 E2 as bait identified multiple possible partner proteins for E2 function (Boner & Morgan 2002) including the DNA replication/repair protein TopBPl. E2 molecules from HPVI6 and HPV18 interact with multiple proteins involved in the cellular response to DNA damage (e.g. p53, BRCAl, PARP and TopBPl). The role of TopBPl in E2 function and the functional response of E2 to DNA damage stimuli were investigated. Overexpression of TopBPl enhances the transcription and replication activation functions of E2. Overexpression of an amino-terminal truncation mutant of TopBPl has no effect on the transcription/replication functions of E2. During this study a novel method for the detection of E1/E2 DNA replication function by real-time PCR was developed. The UVB irradiation of cells resulted in the significant reduction of both E2 transactivation function and E2 protein amount. These results demonstrated that E2 function is altered by cellular DNA damage response proteins and signaling pathways. In many HPV induced cancers the HPV genome is either present integrated into the cellular chromosomes or is maintained episomally with large DNA deletions/rearrangements. Therefore HPV genome stability is a significant risk factor for the development of HPV induced cancer. Thus the fidelity of DNA replication activated by the HPV 16 E1/E2 replication factors was investigated on undamaged and UVC damaged templates in a variety of genetic backgrounds. On undamaged DNA templates there were a significant amount of mutations due to DNA deletions/rearrangements and the frequency of mutation increased when the template was damaged. This increase on damaged templates was marked in cells with defects in key DNA replication or repair proteins. These results highlight the instability of HPV16 genome replication. The interaction of E2 with DNA damage response proteins and the reduction of E2 function in response to DNA damage may be an evolutionary response by the virus to ensure genetic integrity and host cell viability.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:398638 |
| Date | January 2003 |
| Creators | Taylor, Ewan R. |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/30727/ |
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