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Investigating the role of human papillomavirus DNA replication in the development of cervical cancer

King, Lauren E. (2011) Investigating the role of human papillomavirus DNA replication in the development of cervical cancer. PhD thesis, University of Glasgow.

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Abstract

Human papillomavirus type 16 (HPV16) is a causative agent of cervical cancer. In many HPV induced cancers the HPV genome is present integrated into the cellular chromosomes. Integration of HPV DNA into that of the host promotes genomic instability and progression to cancer. Factors that promote integration remain to be fully identified. HPV replication during DNA damage is a factor that may promote viral DNA integration. HPV origin replication is investigated in the presence of DNA damaging agents. HPV replication produces double strand DNA breaks in the viral genome and DNA damaging agents such as etoposide will result in double strand breaks in genomic DNA therefore creating substrates for viral integration. HPV origin replication during DNA damage is not inhibited in vivo and in vitro. In contradiction to these results, replication initiated by the SV40 helicase, Large T antigen, is arrested in response to DNA damage and ATR is the candidate kinase for mediating the arrest. In order to carry out replication of the viral origin, the HPV genome encodes for the E1 protein. E1 forms a di-hexameric helicase complex that replicates the viral genome. The failure of E1 to be targeted by ATR/ATM allows HPV replication in the presence of DNA damaging agents. Such replication will result in double strand breaks in the viral genome ultimately promoting viral integration and progression of cervical cancer. Upon activation of the DNA damage response cellular DNA replication is stalled. The targets of the DNA damage pathways at the replication fork that result in the inhibition of DNA replication are not clearly defined. Evidence has shown that the MCM2-7 cellular helicase complex is targeted for phosphorylation by the ATR/ATM kinases and therefore LTAg helicase may also be targeted in a similar manner in response to DNA damage. The results presented in this thesis support the hypothesis that the replicative helicase is a direct target for phosphorylation by the DNA damaging signalling kinases. Using SV40 replication as a model for eukaryotic DNA replication the results in this thesis show that LTAg is targeted for phosphorylation by the DNA damage signalling kinases ATR/ATM in response to DNA damage. Large T Antigen protein levels decrease after prolonged treatment with etoposide suggesting that the degradation may inhibit the replication functions of this viral helicase. To carry out viral replication HPV encodes two proteins, E1 and E2, which interact with cellular factors to replicate the viral genome. E2 forms homodimers and binds to 12bp palindromic sequences adjacent to the viral origin and recruits the viral helicase, E1, to the origin. The regulatory consequences of the E1.E2 interaction have been controversial. The data in this thesis demonstrates the ability of E1 to stabilise E2 increasing E2 half life. Analysis using a mutant E2 that binds weakly to E1 demonstrates that the stabilisation is a consequence of a direct E1.E2 interaction. Furthermore this thesis also demonstrates that the presence of E1 results in the redistribution of HPV16 E2 by enhancing its affinity for chromatin. The E2 protein tethers the viral genome onto the host chromatin during cellular mitosis thereby ensuring the distribution of viral episomes into both daughter cells. The redistribution of E2 onto chromatin suggests a role for E1 in enhancing HPV genome segregation functions by stabilising the association of E2 with mitotic chromosomes. E2 also regulates transcription from the viral genome. The data in this thesis shows that the E1 protein enhances E2 transcription function in a manner that suggests the E1 protein itself can contribute to transcriptional regulation not simply by E2 stabilisation but by a direct stimulation of E2-mediated transcription. E1 regulation of E2 function is again dependent on a direct protein-protein interaction. Taken together these results suggest that co-expression of E1 with E2 can increase E2 stability, enhance its affinity with chromatin and enhance E2-mediated transcription. These consequences are discussed with relation to the virus life cycle.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: papillomavirus, cervical cancer, replication, helicase, stability, E1, E2
Subjects: Q Science > QR Microbiology > QR355 Virology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
College of Medical Veterinary and Life Sciences > School of Veterinary Medicine
Supervisor's Name: Morgan, Prof. Iain
Date of Award: 2011
Depositing User: miss Lauren E King
Unique ID: glathesis:2011-2726
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 11 Oct 2011
Last Modified: 10 Dec 2012 13:59
URI: http://theses.gla.ac.uk/id/eprint/2726

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