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. / Graduation date: 1990
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/38225 |
| Date | 29 January 1990 |
| Creators | Hu, Chin-Hwa |
| Contributors | Pearson, George D. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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