Topoisomerase-mediated poxviral telomere resolution
Poxviral genomes are linear dsDNA molecules with hairpin telomeres. During replication, these telomeres are joined as inverted repeats within concatemer junction intermediates. The concatemers are then resolved into packageable monomers by a resolution process that creates hairpins bearing mismatched bases. The factors involved in this resolution process are not clear. Recently, a recombinant Shope fibroma virus (SFV) type I topoisomerase was shown to resolve SFV cloned replicative concatemer junctions into haripin-ended molecules ' in vitro'. This thesis is an investigation of the structural transitions involved in poxviral telomere resolution, promoted by SFV topoisomerase. Recombinant SFV topoisomerase resolution was shown to require plasmid substrates bearing inverted repeat telomeric concatemer junctions extruded as cruciforms. The Mg2+-inhibited reaction was slow and inefficient. A pair of 5'TCCTT3' topoisomerase recognition sites, 90 bp 5' of the inverted repeat axis, and near the base of the extruded cruciform, were identified which may be the topoisomerase resolution targets. We suggest that this resolution event involves free branch migration of the cruciform base (Holliday junction) towards the 3' end of resolution targets, covalently-bound by topoisomerase.