Chapter 5 : Cre Recombinase

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The use of Cre recombinase to carry out conditional mutagenesis of transgenes and insert DNA cassettes into eukaryotic chromosomes is widespread ( ). Indeed, a PubMed search for “cre recombinase” in early 2014 returned over 4000 articles. In addition to the numerous and applications that have been reported since Cre was first shown to function in yeast and mammalian cells nearly 30 years ago ( ), the Cre– system has also played an important role in understanding the mechanism of recombination by the tyrosine recombinase family of site-specific recombinases( ). The simplicity of this system, requiring only a single recombinase enzyme and short recombination sequences for robust activity in a variety of contexts ( ), has been an important factor in both cases. Cre has also been used in experiments designed to understand the functions of other recombination systems ( ).

Citation: van Duyne G. 2015. Cre Recombinase, p 119-138. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0014-2014
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Image of Figure 1
Figure 1

The Cre– site-specific recombination reaction. Cre subunits are represented by semi-transparent spheres, with the N-terminal domains (NTDs) in the foreground and the C-terminal domains (CTDs) below. The sites are drawn as observed in crystal structures of intermediates in the Cre– recombination pathway, with the scissile phosphates drawn as yellow spheres and covalently linked phosphotyrosines as cyan spheres. Two Cre subunits bind cooperatively to the substrate sites (Sub1 and Sub2). The structural details of the bending that occurs in the sites at this stage of the reaction are not yet known. The Cre-bound sites associate to form an antiparallel synaptic complex where the bottom (red) strands of are positioned for cleavage (BS-synapse). Cleavage by Tyr324 in the Cre subunits bound to the right half-sites (R) results in formation of covalent 3′-phosphotyrosine linkages to the bottom strands and release of 5′-hydroxyl ends (BS covalent). Exchange of strands between the two sites positions the 5′-hydroxyl groups for attack of the phosphotyrosine linkages on the partner sites, resulting in formation of a four-way Holliday junction intermediate (BS-HJ). Isomerization of BS-HJ to TS-HJ results in activation of the Cre subunits bound to the left half-sites (L), where the top strands (black) are now positioned for cleavage. Cleavage of TS-HJ to form the top-strand covalent intermediate (TS-covalent) and strand exchange to form the top-strand active synapse (TS-synapse) completes the reaction. Dissociation of TS-synapse gives the product sites (Prod1 and Prod2). The structure of TS-synaptic is not currently known; a likely candidate is drawn here (discussed in text).

Citation: van Duyne G. 2015. Cre Recombinase, p 119-138. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0014-2014
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Image of Figure 2
Figure 2

Cre binding to . (A) Cre bound to a half-site. The two domains of Cre form a “C-shaped clamp” that wraps around the DNA duplex. Helices B and D from the N-terminal domain (NTD) straddle the major groove from one face and helix J sits in the major groove from the opposite face. An asterisk marks the point of insertion of several basic residue side chains (not shown) into the minor groove at the end of the site. (B) Cre bound to the site. The site is composed of two 14-bp recombinase-binding elements (RBEs) arranged as inverted repeats around an asymmetric 6-bp crossover region. The RBEs differ only in the base pairs adjacent to the crossover region. Arrows indicate the cleavage sites. Two primary sources of Cre–Cre interactions on the site are evident: the two NTDs interact via helices A and E and the two C-terminal domains interact where helix-N from one subunit is buried in a hydrophobic pocket of the adjacent subunit. The scissile phosphates are drawn as yellow spheres in (A) and (B) and Tyr324 as yellow sticks in (A).

Citation: van Duyne G. 2015. Cre Recombinase, p 119-138. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0014-2014
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Image of Figure 3
Figure 3

Cre- specificity. Polar interactions between helix J and the site are shown. The key interaction is between Arg259 and N7/O6 of G10 in the site. The Glu262 side chain carboxyl makes a water-mediated interaction to N4 of C9 as well as an unusual hydrogen bond to a backbone phosphate (implying a neutral carboxyl side chain). Ser257 also makes a backbone hydrogen bond. The sequence is shown for reference, with the conventional site numbering used by most researchers.

Citation: van Duyne G. 2015. Cre Recombinase, p 119-138. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0014-2014
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Figure 4

The Cre– synaptic complex. The Cre K201A– synaptic complex is shown, with the same DNA coloring as in Fig. 1 , and Cre subunits drawn in semi-transparent space-filling representations. The site is sharply bent in the left half-sites (purple subunits bound) and is undistorted in the right half-sites (green subunits bound). (A) View is from the N-terminal domain face of the complex, with the same orientation as BS-synaptic in Fig. 1 . Substantial interactions between helices A and E are indicated. (B) View is from the opposite face of the complex, where the interlocking interactions formed between helix-N of one subunit and the C-terminal domain of the adjacent subunit are emphasized. Note that the same interactions responsible for cooperative binding in Fig. 2B are responsible for stabilizing the synaptic complex shown here.

Citation: van Duyne G. 2015. Cre Recombinase, p 119-138. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0014-2014
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Figure 5

The Cre active site. Hydrogen-bonding interactions between the seven active site residues and the scissile phosphate are shown for the precleavage intermediate, transition state mimic, and covalent intermediate, based on crystal structures of the corresponding complexes. Glu176 forms additional hydrogen bonds to the backbone amides of both Arg173 and Lys201 in each case, which are not shown. The 5′-hydroxyl group is not present in the covalent intermediate structure, but hydrogen bonding to Lys201/Arg173 is likely (not shown). The cleavage reaction proceeds from left to right, whereas the ligation reaction proceeds right to left in this scheme. Note that O1 and O2 of the transition state are labeled in the opposite sense to that used in Gibb et al. ( ).

Citation: van Duyne G. 2015. Cre Recombinase, p 119-138. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0014-2014
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Image of Figure 6
Figure 6

Schemes to achieve directionality in the Cre reaction. (A) The LE/RE strategy. Recombination between sites with mutated (weakened) right and left half-sites results in a wild-type site and a doubly mutated site, which is a poor substrate. The reverse reaction has reduced efficiency. (B) The recombinase-mediated cassette exchange reaction. The DNA segment to be integrated or exchanged is flanked by incompatible sites in the donor (p-q) and acceptor (r-s) molecules. The sites are incompatible because their crossover sequences differ. Cre-mediated recombination between one site pair results in the integration of the donor (if circular) into the acceptor (the intermediate is not shown). Subsequent recombination between the second pair of sites excises the intervening DNA, resulting in exchange of cassettes between the two molecules.

Citation: van Duyne G. 2015. Cre Recombinase, p 119-138. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0014-2014
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Figure 7

Reconstitution of active Cre from fragments. (A) Cre fragments 1 to 59 and 60 to 341 are inactive individually, but can be re-associated to form an active enzyme if fused to heterodimerizing partners. In the example shown, rapamycin induces dimerization of the FKBP and FRB domains. (B) Overlapping Cre fragments 1 to 196 and 181 to 341 are also inactive individually, but will associate spontaneously to form an active enzyme.

Citation: van Duyne G. 2015. Cre Recombinase, p 119-138. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0014-2014
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Table 1

Selected Cre variants

Citation: van Duyne G. 2015. Cre Recombinase, p 119-138. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0014-2014
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Table 2

Selected variants

Citation: van Duyne G. 2015. Cre Recombinase, p 119-138. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0014-2014

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