Graduate Thesis Or Dissertation
 

Structural requirements for adenovirus DNA replication

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/bk128f37m

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  • The adenovirus replication origins reside in the inverted terminal repetition (ITR) sequences. Replication proceeds unidirectionally by a strand-displacement mechanism from either or both origins to produce duplex daughter DNA plus a displaced parental strand. The displaced strand can be duplicated by two different routes. The cis pathway involves intramolecular hybridization between the inverted terminal repeats to form a panhandle replicative intermediate, where synthesis of the complementary strand is initiated from the newly created duplex origin on the panhandle. Alternatively, complementary strands arising from replication on different molecules can directly hybridize intermolecularly to reform a duplex viral genome. This path is called trans replication. Using plasmid mini-chromosomes as model systems, the structural requirements for adenovirus DNA replication and the relationship between the cis and trans pathways for complementary strand replication was investigated. Plasmids containing single or dual adenovirus origins, with or without inverted repeats, were specially constructed to mimic the structures of the adenovirus genome. Linear plasmids which contain exposed adenovirus origins can engage in adenovirus helper-dependent strand-displacement replication. A special class of panhandle intermediates (foldback structures), arising from the replication of symmetrical dimers and multimers generated by end-to-end ligation during transfection, was identified by two-dimensional gel electrophoresis and physically characterized. Foldback molecules provide the first evidence for the existence of the adenovirus cis replication pathway. Comparing the yields of cis replication products from different plasmids, it is clear that the efficiency of cis replication increases with longer inverted repeats. In addition, work presented here demonstrates that the conversion of displaced strands into duplexes occurs simultaneously and independently by both the cis and the trans pathways. The ability of embedded replication origins to direct adenovirus DNA replication was investigated in transfected cells using the plasmid mini-chromosome model system. Plasmids with origins embedded in circular or linear templates gave rise to replication-proficient linear molecules. Inverted repeats were necessary and sufficient in order to rescue displaced strands from circular or linear templates by the cis pathway. In general, the efficiency of replication on linear templates with embedded origins was higher than on circular templates, but was lower when compared to linear templates with exposed origins. This suggests that the creation of a 3'-end, essential for the cis replication pathway, on displaced strands arising from cicluar molecules may be rate limiting. Alternatively, this might imply that the efficiency of initiation on linear templates is higher than on circular templates. A mutant adenovirus helper was used to investigate the role of homologous recombination in activating plasmids with wild-type adenovirus origin sequences. Recombination between the exposed origins on helper DNA and the embedded origins in linear plasmids might be one possible mechanism to activate embedded origins. However, recombination is clearly not necessary.' Other mechanisms must be involved in the replication of templates which contain embedded origins. The production of tandemly repeated multimers from circular templates suggests that randomly initiated displacement reactions might proceeed continuously around the circles. Subsequently, the displaced strands could be converted into replication-proficient molecules by the cis replication pathway.
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