The life cycle of HIV includes integration of the viral DNA into the host cell DNA which is mediated by the integrase enzyme (In) through the formation of a staggered, double-strand break (DSB) in the host DNA and covalent attachment of the viral DNA ends. We are developing yeast-based genetic detection systems to investigate in vivo mechanisms of action of integrase as well as means for altering the integrase reaction. The consequences of over-expression of two integrases, avian and HIV, are being examined in Saccharomyces cerevisae wild-type and mutant strains defective in recombination and DSB repair. The genetic assays include i) loss of a """"""""target"""""""" plasmid, ii) increased recombination between heteroalleles on a plasmid, and iii) sensitivity to various DNA damaging treatments such as UV, gamma radiation and MMS upon expression of the enzyme. The work with the avian integrase, RAV In, is being done in collaboration with Dr. A.M. Skalka, who had preliminary results with yeast. Our initial work with the RAV In, has demonstrated that the expressed protein could inhibit cell growth when there was a plasmid containing the RAV LTR sequence. We are investigating the requirements for inhibition of cell growth and possible components that could modify this response. We are currently creating new constructs which will allow us to target HIV In to a specific region of a plasmid. This work builds on the efforts of others who showed that a fusion protein of lambda repressor and HIV In can be targeted to lambda operator sites in vitro. We are creating a similar fusion protein for expression in yeast and expect that cutting by the targeted integrase around the lambda operator region placed between two heteroalleles will greatly stimulate recombination. This assay will be used to secreen human cDNAs that affect the In reaction in yeast. We will also examine yeast mutants and drugs which simulate or repress the recombination reaction.
