Summary of Work: An obligate step in the life cycle of HIV is the integration of the viral DNA into the host chromosome. This reaction is mediated by the integrase enzyme (In) through the formation of a staggered double strand break (DSB) in the host DNA and covalent linkage of the viral DNA ends to the host DNA. We are developing yeast based genetic detection systems to investigate in-vivo mechanisms of In, as well as means of altering the reaction, since yeast has repair and recombination systems that can identify and process DNA lesions including DNA breaks. We have examined the effects of overexpression of HIV In. In wildtype and various mutant yeast strains there was no change in growth, morphology, viability, or increased sensitivity to various DNA damaging agents. The lack of a phenotype when overexpressing our HIV In isolate has prompted us to obtain a clinical isolate of the HIV integrase. This gene is currently being cloned under the control of the Gal 1 promoter, allowing us to vary expression of the In from over a 1000 fold range by placing the cells on media containing different amounts of galactose. To increase the sensitivity of our assays we have constructed a targeted HIV In fusion protein. We are currently testing a plasmid construct with three repeats of the target sequence placed between two ura3 heteroalleles that should enable genetic detection of cutting by In. Breaks should stimulate recombination and this will be detected by the appearance of Ura+ cells. Using a system to generate double strand breaks using in-vivo by expression of a restriction enzyme (EcoRI, or HO) we have found that some yeast strains are sensitized to DSBs. These include the rad9 and rad17 checkpoint mutants and mutants for genes involved in Ku mediated end-joining. Since integrase is also proposed to induce DNA breaks, we have initiated investigations of the effects of HIV In in these strains.