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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 now used in research as a means for genome editing.^[1]

You may include any references to papers as in: the use of JSmol in Proteopedia ^[2] or to the article describing Jmol ^[3] to the rescue.

Function

Reference : Crystal structure of a wild-type Cre recombinase-loxP synapse reveals a novel spacer conformation suggesting an alternative mechanism for DNA cleavage activation.

Sequence used to form Cre/DNA cocrystal loxP^[4]

5' -ATAACTTCGTATAGCATACATTATACGAAGTTAT 3'

3' -TATTGAAGCATATCGTATGTAATATGCTTCAATA 5'

DNA Substrate

5' -TATAACTTCGTATAGCATATGCTATACGAAGTTAT- 3'

3' -TATTGAAGCATATCGTATACGATATGCTTCAATA- 5'

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 subject 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) ^[5]. It is corresponding of a 4-5 helices : bundle of two orthogonally packed alpha-hairpins. It is involved too in the interactions with DNA and proteins. The amino termain domain contains 5 helix.

The other domain is the carboxy terminal domain (130-343). It contains helices and beta sheets. The helical parts in Cre-recombinase represent 52% and the Beta sheets parts represent only 5% of the secondary structure of the protein. This proteins is composed of a catalytic triade (Arg 173, His 289, Arg 292) and interacts with magnesium.

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.^[6]

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References

1. ↑ 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
2. ↑ Hanson, R. M., Prilusky, J., Renjian, Z., Nakane, T. and Sussman, J. L. (2013), JSmol and the Next-Generation Web-Based Representation of 3D Molecular Structure as Applied to Proteopedia. Isr. J. Chem., 53:207-216. doi:http://dx.doi.org/10.1002/ijch.201300024
3. ↑ Herraez A. Biomolecules in the computer: Jmol to the rescue. Biochem Mol Biol Educ. 2006 Jul;34(4):255-61. doi: 10.1002/bmb.2006.494034042644. PMID:21638687 doi:10.1002/bmb.2006.494034042644
4. ↑ 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
5. ↑ 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
6. ↑ 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