Cre-Lox recombination
From Wikipedia, the free encyclopedia
Cre-Lox recombination is a special type of site-specific recombination which was patented in the 1980's by DuPont. Although Prof. Brian Sauer is credited with the discovery of this system, many workers from diverse fields have found useful applications of this recombination system including gene knockout technology and the development of Bacteriophage P1 based vectors - PACs (P1 derived Artifical Chromosomes). Cre-Lox recombination involves the targeting of a specific sequence of DNA and splicing it with the help of an enzyme called Cre recombinase.
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[edit] Overview
The Cre/lox system is used as a genetic tool to control site specific recombination events in genomic DNA. This system has allowed researchers to manipulate a variety of genetically modified organisms to control gene expression, delete undesired DNA sequences and modify chromosome architecture.
The system begins with the Cre protein, a site-specific DNA recombinase. Cre can catalyse the recombination of DNA between specific sites in a DNA molecule. These sites, known as loxP sequences, contain specific binding sites for Cre that surround a directional core sequence where recombination can occur.
When cells that have loxP sites in their genome express Cre, a reciprocal recombination event will occur between the loxP sites. The double stranded DNA is cut at both loxP sites by the Cre protein and then ligated (glued) back together. It is a quick and efficient process. The effect of recombination depends on the orientation of the loxP sites. For two lox sites on the same chromosome arm, inverted loxP sites will cause an inversion, while a direct repeat of loxP sites will cause a deletion event. If loxP sites are on different chromosomes it is possible for translocation events to be catalysed by Cre induced recombination.
[edit] Cre recombinase
The Cre (Cyclization Recombination) protein consists of 4 subunits and two domains : The larger carboxyl (C-terminal) domain, and smaller amino (N-terminal) domain. The total protein has 341 amino acids. The C domain is similar in structure to the domain in the Integrase family of enzymes isolated from Bacteriophage λ. This is also the catalytic site of the enzyme.
[edit] Lox P site
Lox P (locus of X-over P1) is a site on the Bateriophage P1 consisting of 34 bp. There exists an asymmetric 8 bp sequence in between with 2 sets of palindromic, 13 bp sequences flanking it. The detailed structure is given below.
| 13bp | 8bp | 13bp |
| ATAACTTCGTATA - | GCATACAT | -TATACGAAGTTAT |
[edit] Holliday Junctions and Homologous Recombination
During genetic recombination, a Holliday junction is formed between the two strands of DNA and a double-stranded break in a DNA molecule leaves a 3’OH end exposed. This reaction is aided with the endonuclease activity of an enzyme. 5’ Phosphate ends are usually the substrates for this reaction, thus extended 3’ regions remain. This 3’ OH group is highly unstable, and the strand on which it is present must find its complement. Since Homologous Recombination occurs after DNA replication, two strands of DNA are available and thus, the 3’ OH group must pair with its complement, and it does so, with an intact strand on the other duplex. Now, one point of crossover has occurred and this is what is called a Holliday Intermediate.
The 3’OH end is elongated (that is, bases are added) with the help of DNA Polymerase. The pairing of opposite strands is what constitutes the crossing-over or Recombination event, which is common to all living organisms, since the genetic material on one strand of one duplex has paired with one strand of another duplex, and has been elongated by DNA polymerase. Further cleavage of Holliday Intermediates results in formation of Hybrid DNA.
This further cleavage or ‘resolvation’ is done by a special group of enzymes called Resolvases. RuvC is just one of these Resolvases which has been isolated in bacteria and yeast.
For many years, it was thought that when the Holliday junction intermediate was formed, the branch point of the junction (where the strands cross over) would be located at the first cleavage site. Migration of the branch point to the second cleavage site would then somehow trigger the second half of the pathway. This model conveniently explained the strict requirement for homology between recombining sites, since branch migration would stall at a mismatch and would not allow the second strand exchange to occur. In more recent years, however, this view has been challenged and most of the current models for Int, Xer and Flp recombination involve only limited branch migration 1–3 base pairs) of the Holliday intermediate, coupled to an isomerisation event that is responsible for switching the strand cleavage specificity.
[edit] Site Specific Recombination: An Overview
As mentioned earlier, the Cre/Lox Recombination system is a type of Site-Specific Recombination. Site-Specific Recombination (SSR) involves as the name suggests, 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. These systems generally 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, cleavage occurs, causing the Recombinase to covalently bond to the DNA at the cleavage site, with the help of a Phospho-Tyrosine bond. As illustrated in the above figure, the Tyrosine residue on the recombinase enzyme gets phosphorylated after the DNA strand is cleaved, leaving a free 5’ Phosphate group to bond with the Tyrosine.
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 synthesises 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. In such a state the virus is called a Prophage. They do so by the process of 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. These are thus site-specific, occurring only at the respective att sites. The Integrase class of enzymes catalyse this particular reaction.
[edit] The Cre-lox system in action
In initial studies in the Bacteriophage P1 system, it was noticed that the gene to be excised was flanked (sandwiched ) by two loxP DNA sequences or sites. The presence of the Cre enzyme greatly resulted in the Phage P1 chromosome assuming a structure that allowed the two loxP sites to come in such close contact that the site-specific recombination mechanism (described above) was capable of taking place. This site-specific recombination resulted in the excision of the flanked gene or DNA sequence from the P1 chromosome into a circular structure.
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