Interaction of the bacteriophage P1 recombinase Cre with the recombining site loxP.

Abstract

The interaction between the P1 recombinase protein Cre and the DNA site at which it acts, loxP, was studied by using nuclease protection techniques. The region of DNA protected by Cre against nuclease attack by DNase I or neocarzinostatin is a 34-base-pair (bp) region containing two 13-bp inverted repeats separated by an 8-bp spacer region. These protected sequences were previously shown to be required for efficient Cre-mediated recombination at loxP. The results of the above protection experiments suggest that no more than 34 bp may be required for loxP recombination and that the asymmetry of loxP recombination is due to the 8-bp spacer sequence. With neocarzinostatin, a specific nucleotide within the 8-bp spacer region is not protected. This nucleotide is located in a 2-bp sequence shown to be involved in a loxP crossover event, suggesting that this region remains exposed after Cre binding. Protection experiments were also done with loxP sites that have the left or right inverted repeat deleted. The nuclease protection pattern of these sites reveals that each loxP site consists of 2 binding domains for Cre, each being composed of one 13-bp inverted repeat and the contiguous 4 bp of the 8-bp spacer region.