Site-specific recombination in mammalian cells catalyzed by gammadelta resolvase mutants: implications for the topology of episomal DNA.
We have transferred the prokaryotic gammadelta 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 the 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.
Schwikardi
M
M
Institute of Genetics, University of Cologne, Weyertal 121, D-50931, Cologne, Germany.
Dröge
P
P
D000818:Animals; D001287:Attachment Sites, Microbiological / Q000235:genetics; D016466:CHO Cells; D002384:Catalysis; D006224:Cricetinae; D004278:DNA, Superhelical / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D045522:Recombinases; D011995:Recombination, Genetic / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D014162:Transfection; D019895:Transposases / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins
D004278:DNA, Superhelical; D045522:Recombinases; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases; D019895:Transposases