Cre-lox recombination in Escherichia coli cells. Mechanistic differences from the in vitro reaction.
The mechanism of the Cre recombinase of bacteriophage P1 in Escherichia coli cells was analyzed by topological methods in order to determine the important features of the in vivo reaction. Lambda infection was used to introduce the cre gene into cells containing plasmid substrates. The products of Cre resolution on substrates with directly repeated sites were predominantly free circles, even though decatenation by DNA gyrase was blocked by the drug norfloxacin. Recombination by Cre was greatly stimulated by negative supercoiling, and inversion occurred inefficiently. These results are strikingly different from those found with purified enzyme in vitro. Our data imply that Cre recombination in vivo is much more tightly controlled than it is in vitro, and that Cre acts predominantly as a resolvase in vivo. We suggest a role for Cre-mediated recombination in P1 plasmid amplification that is consistent with the selectivity of the enzyme in vivo.
Adams
D E
DE
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Bliska
J B
JB
Cozzarelli
N R
NR
D007446:Chromosome Inversion; D003090:Coliphages / Q000201:enzymology / Q000235:genetics*; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005784:Gene Amplification; D005814:Genes, Viral; D005838:Genotype; D019426:Integrases; D007700:Kinetics; D008957:Models, Genetic; D009713:Nucleotidyltransferases / Q000378:metabolism; D010957:Plasmids; D011995:Recombination, Genetic; D015183:Restriction Mapping; D059005:Topoisomerase II Inhibitors; D019895:Transposases; D014764:Viral Proteins; D015678:Viral Structural Proteins / Q000235:genetics*
D004278:DNA, Superhelical; D059005:Topoisomerase II Inhibitors; D014764:Viral Proteins; D015678:Viral Structural Proteins; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; D019426:Integrases; D009713:Nucleotidyltransferases; D019895:Transposases