Cre recombinase

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Cre recombinase
CreRecImage(DNASUBSTRATE).png
Structure of a Cre recombinase enzyme (dimer) bound to its substrate DNA
Identifiers
Organism Enterobacteria phage P1
Symbolcre
Entrez 2777477
RefSeq (Prot) YP_006472.1
UniProt P06956
Other data
EC number 2.7.7.-
Chromosome genome: 0 - 0 Mb
Search for
Structures Swiss-model
Domains InterPro

Cre recombinase is a tyrosine recombinase enzyme derived from the P1 bacteriophage. The enzyme uses a topoisomerase I-like mechanism to carry out site specific recombination events. The enzyme (38kDa) is a member of the integrase family of site specific recombinase and it is known to catalyse the site specific recombination event between two DNA recognition sites (LoxP sites). This 34 base pair (bp) loxP recognition site consists of two 13 bp palindromic sequences which flank an 8bp spacer region. The products of Cre-mediated recombination at loxP sites are dependent upon the location and relative orientation of the loxP sites. Two separate DNA species both containing loxP sites can undergo fusion as the result of Cre mediated recombination. DNA sequences found between two loxP sites are said to be "floxed". In this case the products of Cre mediated recombination depends upon the orientation of the loxP sites. DNA found between two loxP sites oriented in the same direction will be excised as a circular loop of DNA whilst intervening DNA between two loxP sites that are opposingly orientated will be inverted. [1] The enzyme requires no additional cofactors (such as ATP) or accessory proteins for its function. [2]

Contents

The enzyme plays important roles in the life cycle of the P1 bacteriophage, such as cyclization of the linear genome and resolution of dimeric chromosomes that form after DNA replication. [3]

Cre recombinase is a widely used tool in the field of molecular biology. The enzyme's unique and specific recombination system is exploited to manipulate genes and chromosomes in a huge range of research, such as gene knock out or knock in studies. The enzyme's ability to operate efficiently in a wide range of cellular environments (including mammals, plants, bacteria, and yeast) enables the Cre-Lox recombination system to be used in a vast number of organisms, making it a particularly useful tool in scientific research. [4]

Discovery

Studies carried out in 1981 by Sternberg and Hamilton demonstrated that the bacteriophage 'P1' had a unique site specific recombination system. EcoRI fragments of the P1 bacteriophage genome were generated and cloned into lambda vectors. A 6.5kb EcoRI fragment (Fragment 7) was found to permit efficient recombination events. [5] The mechanism of these recombination events was known to be unique as they occurred in the absence of bacterial RecA and RecBCD proteins. The components of this recombination system were elucidated using deletion mutagenesis studies. These studies showed that a P1 gene product and a recombination site were both required for efficient recombination events to occur. The P1 gene product was named Cre (causes recombination) and the recombination site was named loxP (locus of crossing (x) over, P1). [5] The Cre protein was purified in 1983 and was found to be a 35,000 Da protein. [2] No high energy cofactors such as ATP or accessory proteins are required for the recombinase activity of the purified protein. [2] Early studies also demonstrated that Cre binds to non specific DNA sequences whilst having a 20 fold higher affinity for loxP sequences and results of early DNA footprinting studies also suggested that Cre molecules bind loxP sites as dimers. [2]

Cartoon model of Cre recombinase bound to its substrate (DNA). The amino terminal domain is shown in blue whilst the carboxyl domain is green. (A side view) CreRecom+DNA(sideview).png
Cartoon model of Cre recombinase bound to its substrate (DNA). The amino terminal domain is shown in blue whilst the carboxyl domain is green. (A side view)
Cartoon model of Cre recombinase bound to its substrate (DNA). The amino terminal domain is shown in blue whilst the carboxyl domain is green. (A head on view) CreRecom+DNA(head on).png
Cartoon model of Cre recombinase bound to its substrate (DNA). The amino terminal domain is shown in blue whilst the carboxyl domain is green. (A head on view)
Tyrosine recombinase family members [3]
S.cerevisiae Flp recombinase
Bacterial XerC recombinase
Bacterial XerD recombinase
λ integrase protein
HP1 integrase protein

Structure

Cre recombinase consists of 343 amino acids that form two distinct domains. The amino terminal domain encompasses residues 20–129 and this domain contains 5 alpha helical segments linked by a series of short loops. Helices A & E are involved in the formation of the recombinase tetramer with the C terminus region of helix E known to form contacts with the C terminal domain of adjacent subunits. Helices B & D form direct contacts with the major groove of the loxP DNA. These two helices are thought to make three direct contacts to DNA bases at the loxP site. The carboxy terminal domain of the enzyme consists of amino acids 132–341 and it harbours the active site of the enzyme. The overall structure of this domain shares a great deal of structural resemblance to the catalytic domain of other enzymes of the same family such as λ Integrase and HP1 Integrase. This domain is predominantly helical in structure with 9 distinct helices (F−N). The terminal helix (N) protrudes from the main body of the carboxy domain and this helix is reputed to play a role in mediating interactions with other subunits. Crystal structures demonstrate that this terminal N helix buries its hydrophobic surface into an acceptor pocket of an adjacent Cre subunit. [6]

The effect of the two-domain structure is to form a C-shaped clamp that grasps the DNA from opposite sides. [3]

Active site

This cartoon model of Cre recombinase bound to its substrate (DNA) shows the amino acids involved in the active site in red and labelled. This image is generated following cleavage of the DNA. CreRecombActiveSite.png
This cartoon model of Cre recombinase bound to its substrate (DNA) shows the amino acids involved in the active site in red and labelled. This image is generated following cleavage of the DNA.

The active site of the Cre enzyme consists of the conserved catalytic triad residues Arg 173, His 289, Arg 292 as well as the conserved nucleophilic residues Tyr 324 and Trp 315. Unlike some recombinase enzymes such as Flp recombinase, Cre does not form a shared active site between separate subunits and all the residues that contribute to the active site are found on a single subunit. Consequently, when two Cre molecules bind at a single loxP site two active sites are present. Cre mediated recombination requires the formation of a synapse in which two Cre-LoxP complexes associate to form what is known as the synapse tetramer in which 4 distinct active sites are present. [6] Tyr 324 acts as a nucleophile to form a covalent 3’-phosphotyrosine linkage to the DNA substrate. The scissile phosphate (phosphate targeted for nucleophilic attack at the cleavage site) is coordinated by the side chains of the 3 amino acid residues of the catalytic triad (Arg 173, His 289 & Trp 315). The indole nitrogen of tryptophan 315 also forms a hydrogen bond to this scissile phosphate. (n.b A Histidine occupies this site in other tyrosine recombinase family members and performs the same function). This reaction cleaves the DNA and frees a 5’ hydroxyl group. This process occurs in the active site of two out of the four recombinase subunits present at the synapse tetramer. If the 5’ hydroxyl groups attack the 3’-phosphotyrosine linkage one pair of the DNA strands will exchange to form a Holliday junction intermediate. [3]

Applications

Role in bacteriophage P1

Cre recombinase plays important roles in the life cycle of the P1 bacteriophage. Upon infection of a cell the Cre-loxP system is used to cause circularization of the P1 DNA. In addition to this Cre is also used to resolve dimeric lysogenic P1 DNA that forms during the cell division of the phage. [7]

Use in research

Inducible Cre activation is achieved using CreER (estrogen receptor) variant, which is only activated after delivery of tamoxifen. [8] This is done through the fusion of a mutated ligand binding domain of the estrogen receptor to the Cre recombinase, resulting in Cre becoming specifically activated by tamoxifen. In the absence of tamoxifen, CreER will result in the shuttling of the mutated recombinase into the cytoplasm. The protein will stay in this location in its inactivated state until tamoxifen is given. Once tamoxifen is introduced, it is metabolized into 4-hydroxytamoxifen, which then binds to the ER and results in the translocation of the CreER into the nucleus, where it is then able to cleave the lox sites. [9] Importantly, sometimes fluorescent reporters can be activated in the absence of tamoxifen, due to leakage of a few Cre recombinase molecules into the nucleus which, in combination with very sensitive reporters, results in unintended cell labelling. [10] CreER(T2) was developed to minimize tamoxifen-independent recombination and maximize tamoxifen-sensitivity.

Improvements

In recent years, Cre recombinase has been improved with conversion to preferred mammalian codons, the removal of reported cryptic splice sites, an altered stop codon, and reduced CpG content to reduce the risk of epigenetic silencing in mammals. [11] A number of mutants with enhanced accuracy have also been identified. [12]

See also

Related Research Articles

<span class="mw-page-title-main">Integrase</span> Class of enzymes

Retroviral integrase (IN) is an enzyme produced by a retrovirus that integrates—forms covalent links between—its genetic information into that of the host cell it infects. Retroviral INs are not to be confused with phage integrases (recombinases) used in biotechnology, such as λ phage integrase, as discussed in site-specific recombination.

DNA gyrase, or simply gyrase, is an enzyme within the class of topoisomerase and is a subclass of Type II topoisomerases that reduces topological strain in an ATP dependent manner while double-stranded DNA is being unwound by elongating RNA-polymerase or by helicase in front of the progressing replication fork. The enzyme causes negative supercoiling of the DNA or relaxes positive supercoils. It does so by looping the template so as to form a crossing, then cutting one of the double helices and passing the other through it before releasing the break, changing the linking number by two in each enzymatic step. This process occurs in bacteria, whose single circular DNA is cut by DNA gyrase and the two ends are then twisted around each other to form supercoils. Gyrase is also found in eukaryotic plastids: it has been found in the apicoplast of the malarial parasite Plasmodium falciparum and in chloroplasts of several plants. Bacterial DNA gyrase is the target of many antibiotics, including nalidixic acid, novobiocin, albicidin and ciprofloxacin.

A DNA-binding domain (DBD) is an independently folded protein domain that contains at least one structural motif that recognizes double- or single-stranded DNA. A DBD can recognize a specific DNA sequence or have a general affinity to DNA. Some DNA-binding domains may also include nucleic acids in their folded structure.

Cre-Lox recombination is a site-specific recombinase technology, used to carry out deletions, insertions, translocations and inversions at specific sites in the DNA of cells. It allows the DNA modification to be targeted to a specific cell type or be triggered by a specific external stimulus. It is implemented both in eukaryotic and prokaryotic systems. The Cre-lox recombination system has been particularly useful to help neuroscientists to study the brain in which complex cell types and neural circuits come together to generate cognition and behaviors. NIH Blueprint for Neuroscience Research has created several hundreds of Cre driver mouse lines which are currently used by the worldwide neuroscience community.

Site-specific recombinase technologies are genome engineering tools that depend on recombinase enzymes to replace targeted sections of DNA.

Recombinases are genetic recombination enzymes.

<span class="mw-page-title-main">FLP-FRT recombination</span>

In genetics, Flp-FRT recombination is a site-directed recombination technology, increasingly used to manipulate an organism's DNA under controlled conditions in vivo. It is analogous to Cre-lox recombination but involves the recombination of sequences between short flippase recognition target (FRT) sites by the recombinase flippase (Flp) derived from the 2 µ plasmid of baker's yeast Saccharomyces cerevisiae.

Hin recombinase is a 21kD protein composed of 198 amino acids that is found in the bacteria Salmonella. Hin belongs to the serine recombinase family (B2) of DNA invertases in which it relies on the active site serine to initiate DNA cleavage and recombination. The related protein, gamma-delta resolvase shares high similarity to Hin, of which much structural work has been done, including structures bound to DNA and reaction intermediates. Hin functions to invert a 900 base pair (bp) DNA segment within the salmonella genome that contains a promoter for downstream flagellar genes, fljA and fljB. Inversion of the intervening DNA alternates the direction of the promoter and thereby alternates expression of the flagellar genes. This is advantageous to the bacterium as a means of escape from the host immune response.

P1 is a temperate bacteriophage that infects Escherichia coli and some other bacteria. When undergoing a lysogenic cycle the phage genome exists as a plasmid in the bacterium unlike other phages that integrate into the host DNA. P1 has an icosahedral head containing the DNA attached to a contractile tail with six tail fibers. The P1 phage has gained research interest because it can be used to transfer DNA from one bacterial cell to another in a process known as transduction. As it replicates during its lytic cycle it captures fragments of the host chromosome. If the resulting viral particles are used to infect a different host the captured DNA fragments can be integrated into the new host's genome. This method of in vivo genetic engineering was widely used for many years and is still used today, though to a lesser extent. P1 can also be used to create the P1-derived artificial chromosome cloning vector which can carry relatively large fragments of DNA. P1 encodes a site-specific recombinase, Cre, that is widely used to carry out cell-specific or time-specific DNA recombination by flanking the target DNA with loxP sites.

Site-specific recombination, also known as conservative site-specific recombination, is a type of genetic recombination in which DNA strand exchange takes place between segments possessing at least a certain degree of sequence homology. Enzymes known as site-specific recombinases (SSRs) perform rearrangements of DNA segments by recognizing and binding to short, specific DNA sequences (sites), at which they cleave the DNA backbone, exchange the two DNA helices involved, and rejoin the DNA strands. In some cases the presence of a recombinase enzyme and the recombination sites is sufficient for the reaction to proceed; in other systems a number of accessory proteins and/or accessory sites are required. Many different genome modification strategies, among these recombinase-mediated cassette exchange (RMCE), an advanced approach for the targeted introduction of transcription units into predetermined genomic loci, rely on SSRs.

The Tn3 transposon is a 4957 base pair mobile genetic element, found in prokaryotes. It encodes three proteins:

<span class="mw-page-title-main">Homing endonuclease</span>

The homing endonucleases are a collection of endonucleases encoded either as freestanding genes within introns, as fusions with host proteins, or as self-splicing inteins. They catalyze the hydrolysis of genomic DNA within the cells that synthesize them, but do so at very few, or even singular, locations. Repair of the hydrolyzed DNA by the host cell frequently results in the gene encoding the homing endonuclease having been copied into the cleavage site, hence the term 'homing' to describe the movement of these genes. Homing endonucleases can thereby transmit their genes horizontally within a host population, increasing their allele frequency at greater than Mendelian rates.

Conditional gene knockout is a technique used to eliminate a specific gene in a certain tissue, such as the liver. This technique is useful to study the role of individual genes in living organisms. It differs from traditional gene knockout because it targets specific genes at specific times rather than being deleted from beginning of life. Using the conditional gene knockout technique eliminates many of the side effects from traditional gene knockout. In traditional gene knockout, embryonic death from a gene mutation can occur, and this prevents scientists from studying the gene in adults. Some tissues cannot be studied properly in isolation, so the gene must be inactive in a certain tissue while remaining active in others. With this technology, scientists are able to knockout genes at a specific stage in development and study how the knockout of a gene in one tissue affects the same gene in other tissues.

Tre recombinase is an experimental enzyme that in lab tests has removed DNA inserted by HIV from infected cells. Through selective mutation, Cre recombinase which recognizes loxP sites are modified to identify HIV long terminal repeats (loxLTR) instead. As a result, instead of performing Cre-Lox recombination, the new enzyme performs recombination at HIV provirus sites.

RMCE is a procedure in reverse genetics allowing the systematic, repeated modification of higher eukaryotic genomes by targeted integration, based on the features of site-specific recombination processes (SSRs). For RMCE, this is achieved by the clean exchange of a preexisting gene cassette for an analogous cassette carrying the "gene of interest" (GOI).

A P1-derived artificial chromosome, or PAC, is a DNA construct derived from the DNA of P1 bacteriophages and Bacterial artificial chromosome. It can carry large amounts of other sequences for a variety of bioengineering purposes in bacteria. It is one type of the efficient cloning vector used to clone DNA fragments in Escherichia coli cells.

<span class="mw-page-title-main">HIV integration</span>

AIDS is caused by the human immunodeficiency virus (HIV). Individuals with HIV have what is referred to as a "HIV infection". When infected semen, vaginal secretions, or blood come in contact with the mucous membranes or broken skin of an uninfected person, HIV may be transferred to the uninfected person, causing another infection. Additionally, HIV can also be passed from infected pregnant women to their uninfected baby during pregnancy and/or delivery, or via breastfeeding. As a result of HIV infection, a portion of these individuals will progress and go on to develop clinically significant AIDS.

<span class="mw-page-title-main">Floxing</span> Sandwiching of a DNA sequence between two lox P sites

In genetics, floxing refers to the sandwiching of a DNA sequence between two lox P sites. The terms are constructed upon the phrase "flanking/flanked by LoxP". Recombination between LoxP sites is catalysed by Cre recombinase. Floxing a gene allows it to be deleted, translocated or inverted in a process called Cre-Lox recombination. The floxing of genes is essential in the development of scientific model systems as it allows researchers to have spatial and temporal alteration of gene expression. Moreover, animals such as mice can be used as models to study human disease. Therefore, Cre-lox system can be used in mice to manipulate gene expression in order to study human diseases and drug development. For example, using the Cre-lox system, researchers can study oncogenes and tumor suppressor genes and their role in development and progression of cancer in mice models.

<span class="mw-page-title-main">Excisionase</span>

In molecular biology, excisionase is a bacteriophage protein encoded by the Xis gene. It is involved in excisive recombination by regulating the assembly of the excisive intasome and by inhibiting viral integration. It adopts an unusual winged-helix structure in which two alpha helices are packed against two extended strands. Also present in the structure is a two-stranded anti-parallel beta-sheet, whose strands are connected by a four-residue wing. During interaction with DNA, helix alpha2 is thought to insert into the major groove, while the wing contacts the adjacent minor groove or phosphodiester backbone. The C-terminal region of excisionase is involved in interaction with phage-encoded integrase (Int), and a putative C-terminal alpha helix may fold upon interaction with Int and/or DNA.

Nat L. Sternberg was an American molecular biologist and bacteriophage researcher, particularly known for his work on DNA recombination and the phage P1.

References

  1. ↑ Nagy A (Feb 2000). "Cre recombinase: the universal reagent for genome tailoring". Genesis. 26 (2): 99–109. doi: 10.1002/(SICI)1526-968X(200002)26:2<99::AID-GENE1>3.0.CO;2-B . PMID   10686599.
  2. 1 2 3 4 Abremski K, Hoess R (Feb 1984). "Bacteriophage P1 site-specific recombination. Purification and properties of the Cre recombinase protein". The Journal of Biological Chemistry. 259 (3): 1509–1514. doi: 10.1016/S0021-9258(17)43437-5 . PMID   6319400.
  3. 1 2 3 4 Van Duyne GD (2001). "A structural view of cre-loxp site-specific recombination". Annual Review of Biophysics and Biomolecular Structure. 30: 87–104. doi:10.1146/annurev.biophys.30.1.87. PMID   11340053.
  4. ↑ Ennifar E, Meyer JE, Buchholz F, Stewart AF, Suck D (Sep 2003). "Crystal structure of a wild-type Cre recombinase-loxP synapse reveals a novel spacer conformation suggesting an alternative mechanism for DNA cleavage activation". Nucleic Acids Research. 31 (18): 5449–5460. doi:10.1093/nar/gkg732. PMC   203317 . PMID   12954782.
  5. 1 2 Sternberg N, Hamilton D (Aug 1981). "Bacteriophage P1 site-specific recombination. I. Recombination between loxP sites". Journal of Molecular Biology. 150 (4): 467–486. doi:10.1016/0022-2836(81)90375-2. PMID   6276557.
  6. 1 2 Guo F, Gopaul DN, van Duyne GD (Sep 1997). "Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse". Nature. 389 (6646): 40–46. Bibcode:1997Natur.389...40G. doi:10.1038/37925. PMID   9288963. S2CID   4401434.
  7. ↑ Shaikh AC, Sadowski PD (Feb 1997). "The Cre recombinase cleaves the lox site in trans". The Journal of Biological Chemistry. 272 (9): 5695–5702. doi: 10.1074/jbc.272.9.5695 . PMID   9038180.
  8. ↑ Walrath JC, Hawes JJ, Van Dyke T, Reilly KM (2010). "Genetically engineered mouse models in cancer research". Advances in Cancer Research. 106: 113–64. doi:10.1016/S0065-230X(10)06004-5. ISBN   9780123747716. PMC   3533445 . PMID   20399958.
  9. ↑ Kristianto J, Johnson MG, Zastrow RK, Radcliff AB, Blank RD (June 2017). "Spontaneous recombinase activity of Cre-ERT2 in vivo". Transgenic Research. 26 (3): 411–417. doi:10.1007/s11248-017-0018-1. PMC   9474299 . PMID   28409408. S2CID   4377498.
  10. ↑ Álvarez-Aznar A, Martínez-Corral I, Daubel N, Betsholtz C, Mäkinen T, Gaengel K (February 2020). "T2 lines". Transgenic Research. 29 (1): 53–68. doi:10.1007/s11248-019-00177-8. PMC   7000517 . PMID   31641921.
  11. ↑ Shimshek DR, Kim J, Hübner MR, Spergel DJ, Buchholz F, Casanova E, Stewart AF, Seeburg PH, Sprengel R (Jan 2002). "Codon-improved Cre recombinase (iCre) expression in the mouse". Genesis. 32 (1): 19–26. doi:10.1002/gene.10023. PMID   11835670. S2CID   46000513.
  12. ↑ Eroshenko N, Church GM (Sep 2013). "Mutants of Cre recombinase with improved accuracy". Nature Communications. 4: 2509. Bibcode:2013NatCo...4.2509E. doi:10.1038/ncomms3509. PMC   3972015 . PMID   24056590.
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