Poxviruses provide a unique system for studying the synthesis of DNA. Required enzymes and factors are encoded within the viral genome and DNA synthesis and processing occurs within the cytoplasmic compartment of the cell. Therefore, it has been possible to apply genetic and biochemical approaches to the study of DNA replication. My effort has been towards ascertaining the structure and mode of replication of the poxvirus genome with particular emphasis placed on understanding the processing of the telomere-like hairpin structure and the enzymes involved in its replication. The replication of vaccinia virus proceeds through concatemeric intermediates which are resolved into unit length DNA. Plasmids containing the telomere replicative intermediate were, after transfection into cells infected with vaccinia virus, replicated and resolved into linear minichromosomes with sealed terminal hairpins, providing a system to study the cis acting DNA sequences required for telomere resolution. Mutational analysis has demonstrated that a DNA sequence, highly conserved among poxviruses, as well as the palindromic structure of the concatemer junction, is essential for resolution, and that resolution occurred by conservative strand exchange. A model for resolution involving site-specific recombination and orientated branch migration is consistent with this data. Gene products with activities consistent with processing of replicative intermediates have been identified and isolated as first steps in the biochemical characterization of DNA replication in poxviruses. A procedure has been developed for the generation of viral genomes containing large inserts of foreign DNA by ligation of subgenomic viral DNA fragments in the presence of the insert DNA. These DNA molecules are transfected into cells where they can be packaged into viral particles and subsequently propagated as virus.
