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

We have transferred the prokaryotic gamma delta resolvase system to mammalian cells and present a comparative analysis of recombination by wild-type and 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, We also show that tb efficiency of recombination by the resolvase double mutant 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.