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Cre-DMA intermediate

nest strand cleavage first strand cleavage

Holiday junction intermediate

Cfe-DNA intermediate recomtuned DNA

secoTif strand exchange nest strand cleavage

Cre-DMA intermediate

Holiday junction intermediate

Cfe-DNA intermediate secoTif strand exchange recomtuned DNA

synapsed stibslrate DNA

first strand cleavage

Holiday Junction Mechanism

FIGURE 11-8 Mechanism of site-specific recombination by the Cre recom-binase. (a) The left panel shows the scries of intermediate Cre-DMA structures that re-ffea the sequential "one strand at a time" mechanism of exchange. In each of the panels only the two subunits colored in green are in (tie active conformation. Note that after first strand cleavage, the colors of the subunits switch as the second pair of Cre subunits become active for recombination (Source: From Feng Cuo er al 1997. Structure of Qe re-combtnase completed with DNA. Nature 3B9: Copyright O 1997) (b) The nght panel shows the crystal structure of Cre bound to the HoltoJay junction intermediate (corresponding to the third panel in pad a). Note that the two subuntts colored in green are tn a different conformation than are those colored in purple. The complex, therefore, docs not have fourfold symmetry, nonce, for example, that two of the pain, of adjacent DNA "'arms" in the structure are much closer together than are the other pairs. (Copaul D.N., Cuo F, and Van Duyne C D. 1998 EMBOJ. 17' 4175.) Image prepared with BobSarpt, MdScnpt, and Kasier3L>.

FIGURE 11-8 Mechanism of site-specific recombination by the Cre recom-binase. (a) The left panel shows the scries of intermediate Cre-DMA structures that re-ffea the sequential "one strand at a time" mechanism of exchange. In each of the panels only the two subunits colored in green are in (tie active conformation. Note that after first strand cleavage, the colors of the subunits switch as the second pair of Cre subunits become active for recombination (Source: From Feng Cuo er al 1997. Structure of Qe re-combtnase completed with DNA. Nature 3B9: Copyright O 1997) (b) The nght panel shows the crystal structure of Cre bound to the HoltoJay junction intermediate (corresponding to the third panel in pad a). Note that the two subuntts colored in green are tn a different conformation than are those colored in purple. The complex, therefore, docs not have fourfold symmetry, nonce, for example, that two of the pain, of adjacent DNA "'arms" in the structure are much closer together than are the other pairs. (Copaul D.N., Cuo F, and Van Duyne C D. 1998 EMBOJ. 17' 4175.) Image prepared with BobSarpt, MdScnpt, and Kasier3L>.

Box 11-1 Application of Site-Specific Recombination to Genetic Engineering

Because some site-specific recombination systems are so simple, they have become widely used as toots in experimental genetics. Cre recombinase. and its dose relative FLP recombinase, are both used experimentally to delete genes in eukaryottc organisms (also sec example in Chapter 21)

An example of the usefulness of this strategy becomes clear when we cunsider the following hypothetical example. A researcher is interested in the role of a specific gene in (he development of lung canccr and she wishes to study this process using the mouse as a model organism (see Chapter 21). When tlie gene of interest is disrupted ("knocked out"), however, the mice all die during early embryogenesis. Apparently the gene is required very early in development. How CfflEt its role in lung cancer be studied in the adult animal?

Site-specific recombination can often provide the answer. Using routine methods, researchers can introduce recombination sites recognized by Ge (or FLP) flanking the gene of interest These sites will have no effect on the gene's function, unless the recombinase is also present. Therefore, the Oe protein (or FLP protein) can be introduced into the same organism, under the control of a promoter that can be carefully regulated (see Chapter 17). The mice can therefore be allowed to develop in the absence of the recombinase, but (hen after birth, Cre expression can be "turned on" The presence of the recombinase causes deletion of the gene of interest in this case, tiie propensity of the Cre-treated mice (in which the gene is deleted) for lung cancer can now be compared with their "normal" litter mates, in which the gene of interest is still intact Thus, recombination using Cre allows the potential functions of the genes to be uncovered in different stages of development.

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