565:. 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. 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. CreER(T2) was developed to minimize tamoxifen-independent recombination and maximize tamoxifen-sensitivity.
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215:". 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. The enzyme requires no additional
417:
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
521:
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
469:
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
404:
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
249:
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.
210:
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
322:
or accessory proteins are required for the recombinase activity of the purified protein. 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
547:. 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.
470:
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.
278:. A 6.5kb EcoRI fragment (Fragment 7) was found to permit efficient recombination events. The mechanism of these recombination events was known to be unique as they occurred in the absence of bacterial
433:
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.
1116:
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".
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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
771:"Crystal structure of a wild-type Cre recombinase-loxP synapse reveals a novel spacer conformation suggesting an alternative mechanism for DNA cleavage activation"
82:
346:
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)
418:
other subunits. Crystal structures demonstrate that this terminal N helix buries its hydrophobic surface into an acceptor pocket of an adjacent Cre subunit.
338:
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)
129:
226:
The enzyme plays important roles in the life cycle of the P1 bacteriophage, such as cyclization of the linear genome and resolution of dimeric
241:. The enzyme's unique and specific recombination system is exploited to manipulate genes and chromosomes in a huge range of research, such as
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Guo F, Gopaul DN, van Duyne GD (Sep 1997). "Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse".
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639:
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Sternberg N, Hamilton D (Aug 1981). "Bacteriophage P1 site-specific recombination. I. Recombination between loxP sites".
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Inducible Cre activation is achieved using CreER (estrogen receptor) variant, which is only activated after delivery of
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phosphate (phosphate targeted for nucleophilic attack at the cleavage site) is coordinated by the side chains of the 3
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1). The Cre protein was purified in 1983 and was found to be a 35,000 Da protein. No high energy cofactors such as
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The effect of the two-domain structure is to form a C-shaped clamp that grasps the DNA from opposite sides.
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688:"Bacteriophage P1 site-specific recombination. Purification and properties of the Cre recombinase protein"
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of the enzyme. The overall structure of this domain shares a great deal of structural resemblance to the
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loxP DNA. These two helices are thought to make three direct contacts to DNA bases at the loxP site. The
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1067:Álvarez-Aznar A, Martínez-Corral I, Daubel N, Betsholtz C, Mäkinen T, Gaengel K (February 2020).
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Studies carried out in 1981 by
Sternberg and Hamilton demonstrated that the bacteriophage '
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In recent years, Cre recombinase has been improved with conversion to preferred mammalian
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attack the 3’-phosphotyrosine linkage one pair of the DNA strands will exchange to form a
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proteins. The components of this recombination system were elucidated using deletion
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Van Duyne GD (2001). "A structural view of cre-loxp site-specific recombination".
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Kristianto J, Johnson MG, Zastrow RK, Radcliff AB, Blank RD (June 2017).
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domain of the enzyme consists of amino acids 132–341 and it harbours the
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10.1002/(SICI)1526-968X(200002)26:2<99::AID-GENE1>3.0.CO;2-B
597:. A number of mutants with enhanced accuracy have also been identified.
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to form a covalent 3’-phosphotyrosine linkage to the DNA substrate. The
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Structure of a Cre recombinase enzyme (dimer) bound to its substrate DNA
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202:). This 34 base pair (bp) loxP recognition site consists of two 13 bp
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family of site specific recombinase and it is known to catalyse the
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Ennifar E, Meyer JE, Buchholz F, Stewart AF, Suck D (Sep 2003).
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400:
domain encompasses residues 20–129 and this domain contains 5
640:"Cre recombinase: the universal reagent for genome tailoring"
327:
studies also suggested that Cre molecules bind loxP sites as
953:"Genetically engineered mouse models in cancer research"
306:
combination) and the recombination site was named loxP (
1012:"Spontaneous recombinase activity of Cre-ERT2 in vivo"
736:
Annual Review of
Biophysics and Biomolecular Structure
237:
Cre recombinase is a widely used tool in the field of
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Walrath JC, Hawes JJ, Van Dyke T, Reilly KM (2010).
681:
679:
677:
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1161:"Mutants of Cre recombinase with improved accuracy"
912:"The Cre recombinase cleaves the lox site in trans"
262:' had a unique site specific recombination system.
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206:which flank an 8bp spacer region. The products of
190:events. The enzyme (38 kDa) is a member of the
539:Cre recombinase plays important roles in the
8:
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441:of the Cre enzyme consists of the conserved
223:) or accessory proteins for its function.
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1221:at the U.S. National Library of Medicine
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396:that form two distinct domains. The
353:Tyrosine recombinase family members
1159:Eroshenko N, Church GM (Sep 2013).
916:The Journal of Biological Chemistry
910:Shaikh AC, Sadowski PD (Feb 1997).
692:The Journal of Biological Chemistry
517:to this scissile phosphate. (n.b A
577:, the removal of reported cryptic
266:fragments of the P1 bacteriophage
14:
589:to reduce the risk of epigenetic
686:Abremski K, Hoess R (Feb 1984).
392:Cre recombinase consists of 343
748:10.1146/annurev.biophys.30.1.87
1:
969:10.1016/S0065-230X(10)06004-5
705:10.1016/S0021-9258(17)43437-5
457:292 as well as the conserved
186:-like mechanism to carry out
835:10.1016/0022-2836(81)90375-2
823:Journal of Molecular Biology
16:Genetic recombination enzyme
957:Advances in Cancer Research
371:Bacterial XerD recombinase
366:Bacterial XerC recombinase
196:site specific recombination
188:site specific recombination
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1085:10.1007/s11248-019-00177-8
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208:Cre-mediated recombination
174:is a tyrosine recombinase
18:
1028:10.1007/s11248-017-0018-1
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1223:Medical Subject Headings
535:Role in bacteriophage P1
55:Enterobacteria phage P1
929:10.1074/jbc.272.9.5695
775:Nucleic Acids Research
434:
381:HP1 integrase protein
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339:
1165:Nature Communications
612:FLP-FRT recombination
607:Cre-Lox recombination
557:Cre-Lox recombination
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204:palindromic sequences
21:Cre-Lox recombination
376:λ integrase protein
182:. The enzyme uses a
1239:Genetics techniques
1177:2013NatCo...4.2509E
1073:Transgenic Research
1016:Transgenic Research
873:1997Natur.389...40G
638:Nagy A (Feb 2000).
270:were generated and
1185:10.1038/ncomms3509
1130:10.1002/gene.10023
787:10.1093/nar/gkg732
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781:(18): 5449–5460.
524:Holliday junction
513:315 also forms a
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310:cus of crossing (
239:molecular biology
178:derived from the
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545:P1 bacteriophage
486:residues of the
415:catalytic domain
407:carboxy terminal
361:Flp recombinase
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325:DNA footprinting
230:that form after
180:P1 bacteriophage
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130:genome: 0 - 0 Mb
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184:topoisomerase I
172:Cre recombinase
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569:Improvements
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530:Applications
459:nucleophilic
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359:S.cerevisiae
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620:(in German)
587:CpG content
476:nucleophile
439:active site
425:Active site
411:active site
394:amino acids
300:yclization
288:mutagenesis
228:chromosomes
150:Swiss-model
88:YP_006472.1
45:Identifiers
1233:Categories
1069:"T2 lines"
963:: 113–64.
742:: 87–104.
626:References
583:stop codon
541:life cycle
511:tryptophan
502:315). The
498:289 &
484:amino acid
200:LoxP sites
146:Structures
141:Search for
124:Chromosome
106:Other data
19:See also:
591:silencing
563:tamoxifen
519:Histidine
461:residues
445:residues
388:Structure
254:Discovery
219:(such as
217:cofactors
192:integrase
112:EC number
1203:24056590
1171:: 2509.
1146:46000513
1138:11835670
1103:31641921
1046:28409408
997:20399958
805:12954782
756:11340053
666:10686599
601:See also
507:nitrogen
480:scissile
465:324 and
314:) over,
247:knock in
160:InterPro
50:Organism
1244:Enzymes
1194:3972015
1173:Bibcode
1118:Genesis
1094:7000517
1054:4377498
1037:9474299
988:3533445
938:9038180
897:4401434
889:9288963
869:Bibcode
843:6276557
714:6319400
644:Genesis
595:mammals
543:of the
156:Domains
117:2.7.7.-
95:UniProt
76:2777477
1225:(MeSH)
1201:
1191:
1144:
1136:
1101:
1091:
1052:
1044:
1034:
995:
985:
975:
936:
895:
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861:Nature
841:
803:
796:203317
793:
754:
712:
664:
575:codons
504:indole
329:dimers
284:RecBCD
272:cloned
268:genome
213:floxed
176:enzyme
100:P06956
71:Entrez
62:Symbol
1142:S2CID
1050:S2CID
893:S2CID
494:173,
453:289,
449:173,
274:into
264:EcoRI
1199:PMID
1134:PMID
1099:PMID
1042:PMID
993:PMID
973:ISBN
934:PMID
885:PMID
839:PMID
801:PMID
752:PMID
710:PMID
662:PMID
437:The
282:and
280:RecA
1189:PMC
1181:doi
1126:doi
1089:PMC
1081:doi
1032:PMC
1024:doi
983:PMC
965:doi
961:106
924:doi
920:272
877:doi
865:389
831:doi
827:150
791:PMC
783:doi
744:doi
700:doi
696:259
652:doi
593:in
509:of
500:Trp
496:His
492:Arg
472:Tyr
467:Trp
463:Tyr
455:Arg
451:His
447:Arg
320:ATP
292:Cre
245:or
221:ATP
65:cre
1235::
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722:^
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331:.
308:lo
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