The Cre recombinase enzyme is a tetramer composed of four 38.5Kd monomers. Two monomers each bind cooperatively to one half of the loxP site forming a dimer. Then dimers then dimerize to form the functional Cre recombinase enzyme. The protein-protein interactions between the monomers and dimers bend the DNA into a conformation that favors recombination. After that, the active sites of two monomers opposite of each other within opposite dimers cleave the DNA causing a strand exchange and forming a Holliday Junction. Next, the enzyme goes through an isomerization and cleavage of the uncleaved strands ocurs completing the recombination. Refer to figure 2.
Refer to 1crx chime, buttons must be pushed in order to work properly. If you manipulate the chime refresh the page. All images are shown as monomers or dimers.
The Cre recombinase monomer consists of two domains seperated by a short linker. They are the N-terminal domain comprised of amino acids 20-129 (blue) and the C-terminal domain comprised of amino acids 132-341 (red).
The monomers bind the loxP site at the 15 outermost base pairs encompasing the 13 base pair inverted repeat and the outermost 2 base pairs of the spacer. They form C-shaped clamps around the duplex inserting N-terminal orthogonal helicies in the major grove and the C-terminal helicies and loops interact with the DNA in the major and minor grooves on the adjacent face. The N-terminal domain of the monomer contacts the DNA primarily via the orthagonal B and D helicies which make direct contact at three bases. Helix E is alligned directly over the DNA backbone where it forms 3 electrostatic interactions with phosphates of the bent loxP site in the cleaving subunit. The C-terminal domain interacts with the whole 13 base pairs of the inverted repeat region plus the first two base pairs of the spacer or strand exchange region interacting with the major groove, minor groove and sugar phosphate backbone. Helix J is the only one to contact the major groove and the loops between helicies I and J and J and K. Note the C-shaped clamp structure of the monomer
The active site of the monomer consists of multiple residues in the C-terminal region. It contains five important residues, a catalytic triad made up of Arg173 (orange), His289 (yellow), and Arg292 (green) and nucleophilic Tyr324 (blue) and Trp315 (purple). The amino acids for the catalytic domain come from each monomer and there is no overlap of catalytic domains.
The dimerization and tetramerization of the monomers causes the DNA to be bend at an angle of 102 degrees at the spacer site through protein-protein interactions [3].
The mechanism of recombination begins with binding of the monomers shown above. The monomers dimerize and tetramerize. The tetramer forms a hydrophobic strand exchange cavity in the center which contains the central bases for crossover and is formed partly by helicies L and M.