Poxviruses provide a unique system for studying the replication 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. Our effort has been directed towards ascertaining the structure and mode of replication of the poxvirus genome with particular emphasis placed on understanding the processing of the replicative intermediates including the telomere-like hairpin structure and the enzymes involved in its replication. The replication of vaccinia virus proceeds through concatemeric intermediates that are resolved into unit length DNA molecules. Mutational analysis has demonstrated that a cis acting DNA sequence highly conserved among poxviruses as well as the palindromic structure of the concatemer is essential for resolution and that resolution occurs by a process involving conservative strand exchange. A model for resolution involving site-specific recombination and oriented branch migration is consistent with this data. Our present efforts are directed towards determining the trans acting protein components that participate in resolution. We have identified one open reading frame in vaccinia that encodes for a gene with homology to site-specific recombination proteins. This gene is essential for viral growth, synthesized in small amounts after DNA replication, and myristilated. Presently we are trying to determine the function of this gene during vaccinia virus replication. We have also detected an activity in virion extracts that cleaves and cross-links DNA. This activity is dependent on DNA structure, independent of ATP and metal ion concentrations and is similar to reactions observed for other proteins that process DNA structures. We are attempting to purify and identify the protein responsible for this activity and investigate its role in vaccinia virus DNA processing.
