Sandbox Reserved 1130
From Proteopedia
| This Sandbox is Reserved from 15/12/2015, through 15/06/2016 for use in the course "Structural Biology" taught by Bruno Kieffer at the University of Strasbourg, ESBS. This reservation includes Sandbox Reserved 1120 through Sandbox Reserved 1159. |
To get started:
More help: Help:Editing |
1NZB Cre recombinase-loxP synapse
|
Cre recombinase is a tyrosine recombinase enzyme belonging to the integrase family of recombinases. It catalyses the site-specific recombination between two sequences of DNA called loxP sites. This enzyme was discovered in the P1 bacteriophage and it is currently used in research as a means for genome editing.[1]
Contents |
Function
Cre recombinase is used in research as a homologous recombination tool in mammalian, yeasts, plants and bacteria cells, thanks to loxP sites located on the DNA sequence. In a DNA sequence which contains 2 loxP sites parallelly oriented, Cre recombinase cleaves those loxP sites, which deletes the DNA sequence between the 2 cleavage sites, and in the same time, it gathers the two upstream and downstream DNA segments. If the 2 loxP sites are inverted, this will lead to an inversion of the DNA sequence between the 2 loxP sites. If there is 1 loxP site on a chromosome and another one on a different chromosome, the cleavage will cause a translocation.
Moreover, the Cre-loxP system can be inducible thanks to genetically modified organisms in which a parent only expresses the Cre recombinase and the other parent expresses the gene flanked with the loxP sites. Therefore, the next generation which comes from the two parents will express the Cre recombinase and the gene flanked with the loxP sites, that is to say that parents act as controls for the genome editing and the next generation descended from those parents will be the subjects of interest.
Structural highlights
Structure of Cre recombinase
The Cre recombinase protein contains 343 amino acids and the mass of this proteins is 38,540 Dalton.
There are two different important domains :
10-125 : amino terminal domain = SAM domain like (steril alpha motif) [2]. It is corresponding to a 4-5 helices : bundle of two orthogonally packed alpha-hairpins. It is also involved in the interactions with DNA and proteins. The amino terminal domain contains 5 helix (A, B, C, D and E). The helices C, D and E are organized into an antiparallel bundle.
132-341 : the carboxy terminal domain. It contains helices and beta sheets. In this domain, there is a catalytic triade (Arginine 173, Histidine 289, Arginine 292) with conserved nuleophilic residues (Tyrosine 324 and Tryptophane 315) and interacts with magnesium. All residues of the active site comes form the same subunit. The C-terminal domain is similar to the HP1 integrase catalytic domains and lambda integrase.
The helical parts in Cre-recombinase represent 52% and the Beta sheets parts represent only 5% of the secondary structure of the protein.
Mechanism of homologous recombination by Cre recombinase
The synaptic complex of Cre-loxP recombination contains 4 Cre recombinase subunits gathered in 2 antiparallel dimers. Each dimer interacts with DNA on a loxP site which encompasses an 8 bp spacer region flanked by two palindromic 13 bp sequences. Each subunit of the Cre recombinase contains a catalytic activity. However, a sole subunit in each dimer is catalytically active, that is to say that there are 2 catalytic subunit in the synaptic complex.
The mechanism of recombination by Cre recombinase dimers relies on a first cleavage which occurs on a DNA strand, on the first base pair of the spacer region by a nucleophilic attack of Y324 on the first nucleotide phosphate of the spacer region. The targeted phosphate group is coordinated by interactions with residues of the catalytic triad R173, H289, R292 and W315. This reaction leads to the formation of a 3'-phosphotyrosyl covalent intermediate on each catalytic subunit, and 2 free 3'OH ends. These 3'OH ends act as nucleophiles and therefore attack the 3'-phosphotyrosyl group of the nearby Cre recombinase dimer thanks to the proximity of each dimer, which leads to a Holliday junction intermediate in the synaptic complex, that is to say that a strand exchange occured between the 2 Cre recombinase dimers.
Afterwards, an isomerization occurs on each subunit of the synaptic complex, leading to an exchange of catalytic activity. The 2 subunits which encompassed the catalytic activity become catalytically inactive and those which were catalytically inactive become catalytically active. Moreover, the conformational changes induced by the isomerization lead to a conformational change of DNA, which causes a distortion in a DNA strand. This leads to a second cleavage on this distorted DNA strand by a nuclephilic attack of Y324. Like the first cleavage, this reaction leads to the formation of a 3'-phosphotyrosyl covalent intermediate in each catalytic subunit, and 2 free 3'OH ends. Finally, these 3'OH ends, resulting of the second cleavage, act as nucleophiles and consequently attack the phosphate of the 3'-phosphotyrosyl group on the same Cre recombinase dimer. This leads to another strand exchange. As a consequence, the mechanism of recombination by Cre recombinase leads to a double strand exchange of loxP sites.[3]
References
- ↑ Guo F, Gopaul DN, van Duyne GD. Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse. Nature. 1997 Sep 4;389(6646):40-6. PMID:9288963 doi:10.1038/37925
- ↑ Kim CA, Bowie JU. SAM domains: uniform structure, diversity of function. Trends Biochem Sci. 2003 Dec;28(12):625-8. PMID:14659692 doi:http://dx.doi.org/10.1016/j.tibs.2003.11.001
- ↑ Ennifar E, Meyer JE, Buchholz F, Stewart AF, Suck D. 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 Res. 2003 Sep 15;31(18):5449-60. PMID:12954782
