Directional Resolution of Synthetic Holliday Structures by the Cre Recombinase

Linda Lee, Paul D. Sadowski
2001 Journal of Biological Chemistry  
The Cre recombinase of bacteriophage P1 cleaves its target site, loxP, in a defined order. Recombination is initiated on one pair of strands to form a Holliday intermediate, which is then resolved by cleavage and exchange of the other pair of strands to yield recombinant products. To investigate the influence of the loxP sequence on the directionality of resolution, we constructed synthetic Holliday () structures containing either wild-type or mutant lox sites. We found that Cre preferentially
more » ... esolved the synthetic wild-type structures on a particular pair of strands. The bias in the direction of resolution was dictated by the asymmetric loxP sequence since the resolution bias was abolished with symmetric lox sites. Systematic substitutions of the loxP site revealed that the bases immediately 5 to the scissile phosphodiester bonds were primarily responsible for the directionality of resolution. Interchanging these base pairs was sufficient to reverse the resolution bias. The Cre-lox co-crystal structures show that Lys 86 makes a base-specific contact with guanine immediately 5 to one of the scissile phosphates. Substituting Lys 86 with alanine resulted in a reduction of the resolution bias, indicating that this amino acid is important for establishing the bias in resolution. The Cre recombinase of bacteriophage P1 belongs to the integrase family of conservative site-specific recombinases whose members include the integrase of bacteriophage (1), XerC/XerD of Escherichia coli (2), and the Flp protein of the 2m plasmid (3). Cre assists in the efficient segregation of the low copy P1 plasmid during bacterial cell division by resolving dimeric lysogenic P1 plasmids into monomeric units (4). Cre breaks and rejoins DNA at specific sequences called the loxP sites (Fig. 1a) . The 34-bp 1 loxP site is composed of two 13-bp symmetry elements surrounding an 8-bp asymmetric AT-rich region (5). Since the symmetry elements in loxP have identical sequences, the orientation of the loxP site is dictated by the asymmetric central region. A 6-bp overlap region separates the two cleavage sites. This organization of the recombination site is common to the integrase family members, although the interval of the overlap region varies (1-3). Certain recombinases such as -integrase and XerC/XerD have addi-2 The bottom strand is defined here as the strand with the cleavage site between the GC dinucleotide step at positions 4 and 3.
doi:10.1074/jbc.m103739200 pmid:11406627 fatcat:26fppozyjzh55osifs3nt7owcu