Site-specific recombination in mammalian cells catalyzed by gamma delta resolvase mutants: implications for the topology of episomal DNA

We have transferred the prokaryotic gamma delta resolvase system to mammali an cells and present a comparative analysis of recombination by wild-type a nd two mutant resolvases (E124Q and E102Y/E124Q). Transient co-transfection assays using beta-galactosidase as reporter for recombination reveal that episomal DNA does not contain a significant level of unconstrained negative supercoiling, since only mutant resolvases are recombination-proficient, W e also show that tb efficiency of recombination by the resolvase double mut ant is comparable to that observed with Cre, which indicates that resolvase can be used as a new tool for controlled manipulations of episomal DNAs. ( C) 2000 Federation of European Biochemical Societies.