Mutations in nonconserved domains of Ty3 integrase affect multiple stages of the Ty3 life cycle

Ty3, a retroviruslike element of Saccharomyces cerevisiae, transposes into positions immediately upstream of RNA polymerase III-transcribed genes. The Ty3 integrase (IN) protein is required for integration of the replicated, extrachromosomal Ty3 DNA. In retroviral IN, a conserved core region is suff icient for strand transfer activity. In this study, charged-to-alanine scan ning mutagenesis was used to investigate the roles of the nonconserved amin o- and carboxyl-terminal regions of Ty3 IN. Each of the 20 IN mutants was d efective for transposition, but no mutant was grossly defective for capsid maturation. All mutations affecting steady-state levels of mature IN protei n resulted in reduced levels of replicated DNA, even when polymerase activi ty was not grossly defective as measured by exogenous reverse transcriptase activity assay. Thus, IN could contribute to nonpolymerase functions requi red for DNA production in vivo or to the stability of the DNA product. Seve ral mutations in the carboxyl-terminal domain resulted in relatively low le vels of processed 3' ends of the replicated DNA, suggesting that this domai n may be important for binding of IN to the long terminal repeat. Another c lass of mutants produced wild-type amounts of DNA with correctly processed 3' ends. This class could include mutants affected in nuclear entry and tar get association. Collectively, these mutations demonstrate that in vivo, wi thin the preintegration complex, IN performs a central role in coordinating multiple late stages of the retrotransposition life cycle.