Site-specific recombination in human cells catalyzed by phage lambda integrase mutants

Phage lambda Integrase (Int) is the prototype of the so-called integrase fa mily of conservative site-specific recombinases, which includes Cre and FLP . The natural function of Int is to execute integration and excision of the phage into and out of the Escherichia coli genome, respectively. Ln contra st to Cre and FLP, however, wild-type Int requires accessory proteins and D NA supercoiling of target sites to catalyze recombination. Here, we show th at two mutant Int proteins, Int-h (E174 K) and its derivative Int-h/218 (E1 74 K/E218 K), which do not require accessory factors, are proficient to per form intramolecular integrative and excisive recombination in co-transfecti on assays inside human cells. Intramolecular integrative recombination is a lso detectable by Southern analysis in human reporter cell lines harboring target sites attB and attP as stable genomic sequences. Recombination by wi ld-type Int, however, is not detectable by this method. The latter result i mplies that eukaryotic co-factors, which could functionally replace the pro karyotic ones normally required for wild-type Int, are most Likely not pres ent in human cells. (C) 2000 Academic Press.