Cre-Lox recombination
Encyclopedia
Cre-Lox recombination is a special type of site-specific recombination
developed by Dr. Brian Sauer initially for use in activating gene expression in mammalian cell lines and transgenic mice (DuPont
). Subsequently, the laboratory of Dr. Jamey Marth showed that Cre-Lox recombination could be used to delete loxP-flanked chromosomal DNA sequences at high efficiency in selected cell types of transgenic animals, suggesting this approach as a means to define gene function in specific cell types, indelibly mark progenitors in cell fate determination studies, induce specific chromosomal rearrangements for biological and disease modeling, and determine the roles of early genetic lesions in disease (and phenotype) maintenance.. Shortly thereafter, the laboratory of Dr. Klaus Rajewsky reported the production of pluripotent embryonic stem cells bearing a targeted loxP-flanked (floxed) DNA polymerase gene. Combining these advances in collaboration, the laboratories of Drs. Marth and Rajewsky showed in 1994 that Cre-lox recombination could be used for conditional gene targeting in vivo. This technique continues to be used by hundreds of researchers and laboratories around the world as an essential procedure to discover gene function in normal and disease biology, resulting in numerous important discoveries that would otherwise have not been possible. In 2009, Germany awarded the Max Delbrück medal to Dr. Klaus Rajewsky for his role in developing Cre-Lox recombination. Cre-Lox recombination involves the targeting of a specific sequence of DNA
and splicing it with the help of an enzyme called Cre recombinase
. Because systemic inactivation of many genes further cause embryonic lethality, Cre-Lox recombination is commonly used to circumvent this problem. In addition, Cre–Lox recombination provides the best experimental control that presently exists in transgenic animal modeling to link genotypes (alterations in genomic DNA) to the biological outcomes (phenotypes).
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 Cre
protein is a site-specific DNA recombinase, that is, it 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 recombination event can occur between the loxP sites. The double stranded DNA is cut at both loxP sites by the Cre protein. The strands are then rejoined with DNA ligase
in a quick and efficient process. The result 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 of the intervening DNA, 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.
) domain, and smaller amino (N-terminal
) domain. The total protein has 343 amino acid
s. The C domain is similar in structure to the domain in the Integrase
family of enzymes isolated from lambda phage
. This is also the catalytic site of the enzyme.
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, which 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 that have 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 provided convenient explanation for 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.
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.
Site-specific recombinase technology
Site-specific recombinase technology allows for the manipulation of genetic material in order to explore gene function. The success of the Human Genome Project has made recombinant DNA technology an inevitable next step in molecular biology and genetics. As a mechanism of DNA recombination,...
developed by Dr. Brian Sauer initially for use in activating gene expression in mammalian cell lines and transgenic mice (DuPont
DuPont
E. I. du Pont de Nemours and Company , commonly referred to as DuPont, is an American chemical company that was founded in July 1802 as a gunpowder mill by Eleuthère Irénée du Pont. DuPont was the world's third largest chemical company based on market capitalization and ninth based on revenue in 2009...
). Subsequently, the laboratory of Dr. Jamey Marth showed that Cre-Lox recombination could be used to delete loxP-flanked chromosomal DNA sequences at high efficiency in selected cell types of transgenic animals, suggesting this approach as a means to define gene function in specific cell types, indelibly mark progenitors in cell fate determination studies, induce specific chromosomal rearrangements for biological and disease modeling, and determine the roles of early genetic lesions in disease (and phenotype) maintenance.. Shortly thereafter, the laboratory of Dr. Klaus Rajewsky reported the production of pluripotent embryonic stem cells bearing a targeted loxP-flanked (floxed) DNA polymerase gene. Combining these advances in collaboration, the laboratories of Drs. Marth and Rajewsky showed in 1994 that Cre-lox recombination could be used for conditional gene targeting in vivo. This technique continues to be used by hundreds of researchers and laboratories around the world as an essential procedure to discover gene function in normal and disease biology, resulting in numerous important discoveries that would otherwise have not been possible. In 2009, Germany awarded the Max Delbrück medal to Dr. Klaus Rajewsky for his role in developing Cre-Lox recombination. Cre-Lox recombination involves the targeting of a specific sequence of DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
and splicing it with the help of an enzyme called Cre recombinase
Cre recombinase
Cre recombinase, often abbreviated to Cre, is a Type I topoisomerase from P1 bacteriophage that catalyzes site-specific recombination of DNA between loxP sites. The enzyme does not require any energy cofactors and Cre-mediated recombination quickly reaches equilibrium between substrate and reaction...
. Because systemic inactivation of many genes further cause embryonic lethality, Cre-Lox recombination is commonly used to circumvent this problem. In addition, Cre–Lox recombination provides the best experimental control that presently exists in transgenic animal modeling to link genotypes (alterations in genomic DNA) to the biological outcomes (phenotypes).
Overview
The Cre-lox system is used as a genetic tool to control site specific recombinationGenetic recombination
Genetic recombination is a process by which a molecule of nucleic acid is broken and then joined to a different one. Recombination can occur between similar molecules of DNA, as in homologous recombination, or dissimilar molecules, as in non-homologous end joining. Recombination is a common method...
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 Cre
Cre recombinase
Cre recombinase, often abbreviated to Cre, is a Type I topoisomerase from P1 bacteriophage that catalyzes site-specific recombination of DNA between loxP sites. The enzyme does not require any energy cofactors and Cre-mediated recombination quickly reaches equilibrium between substrate and reaction...
protein is a site-specific DNA recombinase, that is, it 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 recombination event can occur between the loxP sites. The double stranded DNA is cut at both loxP sites by the Cre protein. The strands are then rejoined with DNA ligase
DNA ligase
In molecular biology, DNA ligase is a specific type of enzyme, a ligase, that repairs single-stranded discontinuities in double stranded DNA molecules, in simple words strands that have double-strand break . Purified DNA ligase is used in gene cloning to join DNA molecules together...
in a quick and efficient process. The result 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 of the intervening DNA, while a direct repeat of loxP sites will cause a deletion event. If loxP sites are on different chromosomes it is possible for translocation
Chromosomal translocation
In genetics, a chromosome translocation is a chromosome abnormality caused by rearrangement of parts between nonhomologous chromosomes. A gene fusion may be created when the translocation joins two otherwise separated genes, the occurrence of which is common in cancer. It is detected on...
events to be catalysed by Cre induced recombination.
Cre recombinase
The Cre (causes recombination) protein consists of 4 subunits and two domains: The larger carboxyl (C-terminalC-terminal end
The C-terminus is the end of an amino acid chain , terminated by a free carboxyl group . When the protein is translated from messenger RNA, it is created from N-terminus to C-terminus...
) domain, and smaller amino (N-terminal
N-terminal end
The N-terminus refers to the start of a protein or polypeptide terminated by an amino acid with a free amine group . The convention for writing peptide sequences is to put the N-terminus on the left and write the sequence from N- to C-terminus...
) domain. The total protein has 343 amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
s. The C domain is similar in structure to the domain in the Integrase
Integrase
Retroviral integrase is an enzyme produced by a retrovirus that enables its genetic material to be integrated into the DNA of the infected cell...
family of enzymes isolated from lambda phage
Lambda phage
Enterobacteria phage λ is a temperate bacteriophage that infects Escherichia coli.Lambda phage is a virus particle consisting of a head, containing double-stranded linear DNA as its genetic material, and a tail that can have tail fibers. The phage particle recognizes and binds to its host, E...
. This is also the catalytic site of the enzyme.
Lox P site
Lox P (locus of X-over P1) is a site on the Bacteriophage P1 consisting of 34 bp. There exists an asymmetric 8 bp sequence in between with two sets of palindromic, 13 bp sequences flanking it. The detailed structure is given below.| 13bp | 8bp | 13bp |
| ATAACTTCGTATA - | GCATACAT | -TATACGAAGTTAT |
Holliday junctions and homologous recombination
During genetic recombination, a Holliday junctionHolliday junction
A Holliday junction is a mobile junction between four strands of DNA. The structure is named after Robin Holliday, who proposed it in 1964 to account for a particular type of exchange of genetic information he observed in yeast known as homologous recombination...
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, which 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 that have 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 provided convenient explanation for 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.
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
Integrase
Retroviral integrase is an enzyme produced by a retrovirus that enables its genetic material to be integrated into the DNA of the infected cell...
class of enzymes catalyse this particular reaction.