Graduate Thesis Or Dissertation
 

Sequence conversion during adenovirus DNA replication

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/c534fr22d

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  • The work in this thesis has provided conclusive genetic evidence that "panhandle" intermediates form during adenovirus replication. Adenovirus chromosomes lacking 51 by from their left -hand termini are infectious and capable of regenerating the missing origin sequence. Yet if an entire inverted terminal repeat is removed, the adenovirus chromosome is no longer viable. This first suggested, but did not prove, that "panhandles" formed during adenovirus replication. Homologous recombination or postreplicative overlap recombination could generate the same outcome. Analysis of the segregation of markers in the inverted repeats of adenovirus minichromosomes shows that homologous recombination does not mediate end repair. A special case was also found where postreplicative overlap recombination failed to transfer sequences between the inverted repeats, but similar molecules could exchange sequence information during "panhandle" formation. The exchange of information between inverted repeats is referred to as sequence conversion. A number of length and/or orientation constraints on sequence conversion during adenovirus DNA replication were identified. A length- and orientation-dependent constraint was found for gap filling close to "panhandle" loops. Polymerization towards the loop could occur even when the gap was only 6 by away. In contrast, polymerization away from the "panhandle" loop at a gap at 6 bp, did not take place. This steric constraint could reflect an asymmetry in the action of adenovirus DNA polymerase. A similar length and/or orientation dependent constraint was found for the removal of bulges (3 by and 4 by mismatches). Incision in the bulge of the 5' inverted repeat caused a block to the completion of sequence conversion at that site. When the bulge was in the 3' inverted repeat, a length requirement for successful removal was demonstrated. When 6 by or 39 by separated the bulge from the "panhandle" loop, removal of the bulge was not detected. When the distance was 79 bp, 184 bp, or 217 bp, bulges were successfully removed. The molecular basis for this obstruction remains to be determined. Moreover, incision in bulges located in the 3' inverted repeat triggers directional coconversion. Finally, small loops placed close to the site of polymerization did not cause the same length and orientation dependent constraints as did the "panhandle" loop.
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