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 outside of the nucleus 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. This project is endeavoring to discern the cis-acting and trans-acting components required for the processing of replicative intermediates, an integral process in vaccinia DNA replication. This knowledge will be used for the construction of highly attenuated safe poxvirus vectors and for the evaluation of presently used poxvirus vectors. The replication of vaccinia virus proceeds through concatemeric highly branched 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 of the telomere 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. A separate, sequence-independent mechanism is responsible for the resolution of the numerous branch points present in the replicative intermediates. Our present efforts are directed towards determining the trans-acting protein components that participate in telomeric as well as branch resolution. To investigate the role candidate genes play in vaccinia virus replication we have constructed recombinant viruses in which the endogenous target gene has been replaced with an inducible copy of the gene dependent on isopropyl-b-D-thiogalactopyranoside (IPTG) for expression. The role of the target protein can be investigated by examining the viral life cycle in the presence and absence of the inducible gene product. The genes, A5, which has been shown to co-purify with a virally encoded nuclease activity, and A32, a gene with homology to DNA packaging enzymes, have been investigated in this manner. The A5 protein appears to be required for the condensation of the immature virion to form the brick-shaped mature intracellular mature virion and A32 is needed for incorporation of DNA into the immature virion. Present efforts are directed towards the identification and determination of the role of a nuclease encoded by vaccinia virus. This protein is encoded at late times after infection, is included in the virion particle, and can cleave and cross-link DNA structures which mimic those found in poxvirus DNA replicative intermediates. Preliminary data suggests the gene encoding the protein is in the left hand portion of the viral genome.