The cyclization of linear DNA in Escherichia coli by site-specific recombination.
The efficiency with which linearized plasmid DNA can transform competent Escherichia coli can be significantly increased by use of the Cre-lox site-specific recombination system of phage P1. Linear plasmid molecules containing directly repeated loxP sites (lox2 plasmids) are cyclized in Cre+ E. coli strains after introduction either by transformation or by mini-Mu transduction. Exonuclease V activity of the RecBC enzyme inhibits efficient cyclization of linearized lox2 plasmids after transformation. By use of E. coli mutants which lack exonuclease V activity, Cre-mediated cyclization results in transformation efficiencies for linearized lox2 plasmids identical to those obtained with covalently closed circular plasmid DNA. Moreover, Cre+ E. coli recBC strains allow the efficient recovery of lox2 plasmids integrated within large linear DNA molecules such as the 150-kb genome of pseudorabies virus.
Sauer
B
B
E.I. du Pont de Nemours & Co., Inc., Experimental Station, CR & DD, Wilmington, DE 19880-0328.
Henderson
N
N
D001435:Bacteriophages / Q000235:genetics; D002384:Catalysis; D004254:DNA Nucleotidyltransferases / Q000235:genetics; D004270:DNA, Circular; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics*; D005838:Genotype; D019426:Integrases; D010957:Plasmids; D015183:Restriction Mapping; D014161:Transduction, Genetic; D014170:Transformation, Genetic; D014764:Viral Proteins
D004270:DNA, Circular; D004274:DNA, Recombinant; D014764:Viral Proteins; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; D019426:Integrases