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

The site-specific recombinase Cre must employ control mechanisms to impose directionality on recombination. When two recombination sites (locus of cro ssing 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 inte rvening DNA. If two sites are placed as inverted repeats, the intervening s egment is flipped around. Cre catalyzes these reactions in the absence of p rotein 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 recom bination is altered when the expression of Cre is increased. This leads to deletion instead of inversion on substrates carrying two loxP sites as inve rted repeats. The nucleotide sequence composition of loxP sites remaining i n aberrant products sequence compost indicates that site alignment and/or D NA strand transfer in the in vivo Cre-loxP recombination pathway are not al ways tightly controlled. (C) 2001 Academic Press.