Graduate Thesis Or Dissertation
 

Intramolecular and intermolecular strand hybridization during adenovirus DNA replication

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  • Adenovirus initiates strand-displacement DNA replication from origins located in identical inverted terminal repetitions (ITRs). Panhandle structures, formed by base-pairing between ITRs on the displaced strands, have been proposed as replication intermediates for complementary strand synthesis. Plasmid molecules containing a single adenovirus terminal sequence and specially arranged inverted repeat sequences were used as a model system to study the length and sequence integrity of panhandles. By making a series of unidirectional deletions in the panhandle sequence, it is possible to show that 31 by or longer are sufficient for panhandle formation, but 28 by or smaller are not. Removal of long stretches of unpaired 3' nucleotides distal to the panhandle is extremely efficient and rectification of certain kinds of mismatched sequences in the panhandle region occurs readily. These results argue for the formation of panhandles during adenovirus DNA replication and provide a mechanism for maintaining sequence identity between distantly located inverted repetitions. These size constraints may also explain why the adenovirus ITRs are larger than viral DNA replication origins. Moreover, when multiple copies of the inverted repeat sequences are introduced into the plasmid molecules, it appears that there is no preferential selection of specific pairs of inverted repeats for panhandle formation. Two different pathways have been found to be involved in adenovirus complementary strand replication. Cis replication occurs by intramolecular base-pairing of complementary sequences within a displaced single strand to form a panhandle structure, and transreplication involves intermolecular hybridization of complementary sequences from different displaced single strands to form a heteroduplex to complete the replication cycle. In order to study the relative participation of the cis and trans mechanisms in the adenovirus replication cycle, plasmid molecules were constructed with adenovirus origins and inverted repeat sequences that can pair distinguishably both within and between displaced strands. The results to date demonstrate that both cis and trans pathways function simultaneously during adenovirus DNA replication. Moreover, it appears as little as two copies of a 89-bp complementary sequence support trans replication although a single copy of 89 by does not. Both cis and trans pathways show efficient sequence rectification of terminal non-homology at the 3' ends of strands, a phenomenon not detected in eukaryotic cells before.
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