Cre-Lox Recombination - Site-specific Recombination

Site-specific Recombination

Site-specific recombination (SSR) involves specific sites for the catalysing action of special enzymes called recombinases. Cre, or cyclic recombinase, is one such enzyme. Site-specific recombination is, thus, the enzyme-mediated cleavage and ligation of two defined deoxynucleotide sequences.

A number of conservative site-specific recombination systems have been described in both prokaryotic and eukaryotic organisms. In general, these systems use one or more proteins and act on unique asymmetric DNA sequences. The products of the recombination event depend on the relative orientation of these asymmetric sequences. Many other proteins apart from the Recombinase are involved in regulating the reaction. During site-specific DNA recombination, which brings about genetic rearrangement in processes such as viral integration and excision and chromosomal segregation, these recombinase enzymes recognize specific DNA sequences and catalyse the reciprocal exchange of DNA strands between these sites.

Mechanism of action

Initiation of site-specific recombination begins with the binding of recombination proteins to their respective DNA targets. A separate recombinase recognizes and binds to each of two recombination sites on two different DNA molecules or within the same DNA. At the given specific site on the DNA, the hydroxyl group of the tyrosine attacks a phosphate group in the DNA using a direct transesterification mechanism linking the recombinase protein to the DNA via a phospho-tyrosine linkage. This conserves the energy of the phosphodiester bond, allowing the reaction to be reversed without the involvement of a high-energy cofactor.

Cleavage on the other strand also causes a phospho-tyrosine bond between DNA and the enzyme. At both the DNA duplexes, the bonding of the phosphate group to tyrosine residues leave a 3’ OH group free. In fact, the enzyme-DNA complex is an intermediate stage, which is followed by the ligation of the 3’ OH group of one DNA strand to the 5’ phosphate group of the other DNA strand, which is covalently bonded to the tyrosine residue; that is, the covalent linkage between 5’ end and tyrosine residue is broken. This reaction synthesizes the Holliday Junction discussed earlier.

In this fashion, opposite DNA strands are joined together. Subsequent cleavage and rejoining cause DNA strands to exchange their segments. Protein-Protein interactions drive and direct strand exchange. Energy is not compromised, since the Protein-DNA linkage makes up for the loss of the Phosphodiester bond, which occurred during cleavage.

Site-specific Recombination is also an important process that viruses, such as bacteriophages, adopt to integrate their genetic material into the infected host. The virus, called a prophage in such a state, accomplishes this via integration and excision. The points where the integration and excision reactions occur are called the attachment (att) sites. An attP site on the Phage exchanges segments with an attB site on the Bacterial DNA. Thus, these are site-specific, occurring only at the respective att sites. The integrase class of enzymes catalyse this particular reaction.