All ETDs from UAB

Advisory Committee Chair

Louise T Chow

Advisory Committee Members

William J Britt

Thomas R Broker

Igor Chesnokov

N Patrick Higgins

Kent T Keyser

Document Type

Dissertation

Date of Award

2010

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Human papillomaviruses (HPVs) are small, double-stranded deoxyribonucleic acid (DNA) tumor viruses capable of establishing persistent infections in the epithelia. After infecting actively-dividing basal cells, the papillomavirus (PV) genome is main-tained as extrachromosomal nuclear plasmids. It is largely unknown how the viral ge-nome is maintained in actively-dividing cells. Our lab demonstrated that several HPV genotypes appear to employ a distinct strategy to facilitate partitioning of HPV DNA into daughter cells during mitosis. Association of the HPV-11 origin of replication (ori)-binding protein E2 with the mitotic apparatus via cellular adapter proteins is thought to mediate equal partitioning of HPV genomes into daughter cells. The focus of this disser-tation project was to devise and test a technique allowing direct visualization of repli-cated HPV DNA during mitosis. First, I developed a tracking plasmid system for observing newly-replicated HPV ori-containing plasmids during mitosis. Certain DNA-binding proteins only bind to a unique DNA sequence. I incorporated DNA binding-site arrays into HPV ori-containing plasmids, expressed DNA-binding domain-GFP fusion proteins, and visualized HPV tracking plasmids using high-resolution, high-sensitivity microscopy. Usage of Gal4 DNA-binding site (Gal4-DBD)- and TetR GFP fusion proteins each failed. Alternatively, rtTA3-eGFP-nls facilitated efficient, doxycycline(dox)-inducible, direct, in situ fluores-cent detection of replicated HPV ori-containing plasmid without side-effects. TetO ar-rays I successfully-utilized were smaller than those previously-reported by others. With my 48-copy, tandem TetO array, plasmid was only detectable after exposure of rtTA3-eGFP-nls to dox. To our knowledge this is the first time that DNA has been inducibly-labeled by fluorescence. Secondly, I report that we were able to follow the fate of HPV plasmids during mitosis using imagery. A short, pre-treamtent with dox prior to fixation allowed us to visualize replicated plasmids as small, discrete foci with relatively-uniform intensities. Control TetO array-only control plasmids mostly coalesced to form a small number of large, bright aggregates. We suggest that replicated HPV DNA can persist in dividing cells rather than being sequestered and degraded as was the case with control non-replicated control plasmids.

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