Geometry of the DNA Substrates in Cre-loxP Site-Specific Recombination

Abstract

Cre recombinase is a member of a large family of site-specific recombination enzymes that performs a cut-and-paste operation between two specific DNA sequences. Our goal has been to understand the mechanism of this complex reaction by trapping and characterizing the three-dimensional structures of each of the reaction intermediates. This work has led to high resolution crystallographic models of (i) the initial synaptic complex, (ii) the covalent Cre- DNA intermediate, and (iii) the Holliday junction intermediate. The Cre-loxP system appears to function by creating at the outset a protein-DNA architecture that resembles that of the Holliday junction intermediate that is eventually formed. The “arms” of the loxP sites are initially bent by about 75° in the synaptic complex, forming a nearly planar arrangement that is held fixed, while cleavage and strand exchange occur in the central region between the arms. The simplest view of the recombination pathway is that it contains two symmetrical halves, each of which uses this Holliday junction-like architectural framework to mediate the cleavage and ligation steps. The two halves are linked by a subtle isomerization of the Holliday intermediate that switches the roles of the recombinase subunits and allows exchange of the second pair of DNA strands. In this paper, we summarize recent structural results from our laboratory, with an emphasis on the geometry of the DNA substrates.