Altered directionality in the Cre-LoxP site-specific recombination pathway

• Published in: Journal of molecular biology 2001-08, Vol.311 (3), p.453-459
• Main Authors: Aranda, M, Kanellopoulou, C, Christ, N, Peitz, M, Rajewsky, K, Dröge, P
• Format: Article
• Language: English
• Subjects: Attachment Sites, Microbiological - genetics; Bacteriophage P1 - enzymology; Bacteriophage P1 - genetics; Base Sequence; Blotting, Western; Cre recombinase; Dimerization; Escherichia coli; Escherichia coli - genetics; Genes, Reporter - genetics; Genes, Viral - genetics; Integrases - genetics; Integrases - metabolism; Mutagenesis, Site-Directed - genetics; Recombination, Genetic - genetics; Repetitive Sequences, Nucleic Acid - genetics; Sequence Deletion - genetics; Viral Proteins - genetics; Viral Proteins - metabolism
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.2001.4888

The site-specific recombinase Cre must employ control mechanisms to impose directionality on recombination. When two recombination sites (locus of crossing over in phage P1, loxP) are placed as direct repeats on the same DNA molecule, collision between loxP-bound Cre dimers leads to excision of intervening DNA. If two sites are placed as inverted repeats, the intervening segment is flipped around. Cre catalyzes these reactions in the absence of protein co-factors. Current models suggest that directionality is controlled at two steps in the recombination pathway: the juxtaposition of loxP sites and the single-strand-transfer reactions within the synaptic complex. Here, we show that in Escherichia coli strain 294-Cre, directionality for recombination is altered when the expression of Cre is increased. This leads to deletion instead of inversion on substrates carrying two loxP sites as inverted repeats. The nucleotide sequence composition of loxP sites remaining in aberrant products indicates that site alignment and/or DNA strand transfer in the in vivo Cre-loxP recombination pathway are not always tightly controlled.