DNA Topology and Geometry in Flp and Cre Recombination

Vetcher, Alexandre A.; Lushnikov, Alexander Y.; Navarra-Madsen, Junalyn; Scharein, Robert G.; Lyubchenko, Yuri L.; Darcy, Isabel K.; Levene, Stephen D.

doi:10.1016/j.jmb.2006.01.037

Cited by 57 publications

(54 citation statements)

References 52 publications

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“…This chirality was interpreted as a right-handed crossing of sites in the synaptic complex that is maintained through the reaction to provide recombinant products with the same handedness. More recently, a topological analysis based on atomic force microscopy images following in vitro recombination by the Cre and Flp recombinases has supported the chiral recombination mechanism (11). In the latter work, a model was proposed in which a chiral HJ intermediate is responsible for the biased topological outcomes of recombination.…”

Section: Discussionmentioning

confidence: 94%

“…For example, synaptic -int att and CreloxP complexes have been observed by electron microscopy (7,8) and as slower-migrating bands on non-denaturing polyacrylamide gels (9,10); atomic force microscopy has been used to probe synaptic complexes in the Int, Flp, and Cre systems (11,12); and single molecule light microscopy has recently been used to observe formation of synaptic complexes during -int recombination (13). In addition, crystal structures have been reported for synaptic Cre-DNA complexes (14,15) and for a truncated -int-DNA complex (16).…”

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confidence: 99%

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Synapsis of loxP Sites by Cre Recombinase

Ghosh

Guo²,

Duyne

2007

Journal of Biological Chemistry

131

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Cre recombinase catalyzes site-specific recombination between 34-bp loxP sites in a variety of topological and cellular contexts. An obligatory step in the recombination reaction is the association, or synapsis, of Cre-bound loxP sites to form a tetrameric protein assembly that is competent for strand exchange. Using analytical ultracentrifugation and electrophoresis approaches, we have studied the energetics of Cre-mediated synapsis of loxP sites. We found that synapsis occurs with a high affinity (K d ‫؍‬ 10 nM) and is pH-dependent but does not require divalent cations. Surprisingly, the catalytically inactive Cre K201A mutant is fully competent for synapsis of loxP sites, yet the inactive Y324F and R173K mutants are defective for synapsis. These findings have allowed us to determine the first crystal structures of a pre-cleavage Cre-loxP synaptic complex in a configuration representing the starting point in the recombination pathway. When combined with a quantitative analysis of synapsis using loxP mutants, the structures explain how the central 8 bp of the loxP site are able to dictate the order of strand exchange in the Cre system.Site-specific recombinases from the tyrosine recombinase family are used by bacteria and yeast to mediate the integration, excision, resolution, and inversion of DNA segments to carry out a wide variety of biological functions (1, 2). In the simplest systems, typified by the bacteriophage P1 Cre recombinase (3) and the Saccharomyces cerevisiae Flp recombinase (4), only the recombinase enzymes and DNA substrates containing 34-bp recombination sequences are required for efficient recombination. In others, such as the bacteriophage -integrase (-int) 3 (5) and the Escherichia coli XerC and XerD recombinases (6), efficient recombination requires more complex recombination sites and the activities of auxiliary proteins that tightly regulate the forward and reverse reactions. A remarkable property of both the simple and complex systems is their ability to efficiently synapse (associate) recombining sites that can be located far from one another on the same chromosome or, for bacteriophage integration, are in separate DNA molecules.Synapsis of recombining sites has been visualized in tyrosine recombinase systems using a variety of complementary experimental approaches. For example, synaptic -int att and CreloxP complexes have been observed by electron microscopy (7, 8) and as slower-migrating bands on non-denaturing polyacrylamide gels (9,10

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Section: Discussionmentioning

confidence: 94%

mentioning

confidence: 99%

Synapsis of loxP Sites by Cre Recombinase

Ghosh

Guo²,

Duyne

2007

Journal of Biological Chemistry

131

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“…These recombinases are known to recognize palindromic sequences (3,40). Interestingly, a 14-bp palindromic sequence (CGTTTCTTGAAAAG, where underlining indicates complementary nucleotides) was found also at the nlpC promoter region (Fig.…”

Section: Discussionmentioning

confidence: 97%

FliZ Acts as a Repressor of the ydiV Gene, Which Encodes an Anti-FlhD ₄ C ₂ Factor of the Flagellar Regulon in Salmonella enterica Serovar Typhimurium

2011

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YdiV acts as an anti-FlhD 4 C 2 factor, which negatively regulates the class 2 flagellar operons in poor medium in Salmonella enterica serovar Typhimurium. On the other hand, one of the class 2 flagellar genes, fliZ, encodes a positive regulator of the class 2 operons. In this study, we found that the FliZ-dependent activation of class 2 operon expression was more profound in poor medium than in rich medium and not observed in the ydiV mutant background. Transcription of the ydiV gene was shown to increase in the fliZ mutant. Purified FliZ protein was shown in vitro to bind to the promoter region of the nlpC gene, which is located just upstream of the ydiV gene, and to repress its transcription. These results indicate that FliZ is a repressor of the nlpC-ydiV operon and activates the class 2 operons by repressing ydiV expression. Therefore, the fliZ and ydiV genes form a regulatory loop.Salmonella enterica serovar Typhimurium cells swim by means of rotating flagella through their environment. The individual flagellum consists of three structural parts, a basal body, a hook, and a filament. More than 50 genes are specifically required for flagellar formation and function (1,2,4,26). These genes are organized into at least 15 operons, and their transcriptional expression forms a highly organized threetiered cascade called a flagellar regulon (22,27). The flhDC operon is the sole one belonging to class 1 and encodes FlhD and FlhC, which assemble into an FlhD 4 C 2 heterohexamer acting as an activator of class 2 operons (31, 42). Class 2 contains operons encoding component proteins for the hookbasal body structure and the flagellum-specific type III export apparatus as well as a gene encoding the flagellum-specific sigma factor 28 (FliA), essential for class 3 expression (33). Class 3 operons encode proteins involved in filament assembly and flagellar function.28 activity is negatively controlled by an anti-28 factor, FlgM (15,24,34), which is excreted from the cell through the flagellum-specific type III export apparatus upon completion of hook-basal body assembly (11,19).Class 2 operons are transcribed by 70 RNA polymerase in the presence of the FlhD 4 C 2 complex, which binds to the DNA region upstream of the class 2 promoter (13,30,31). Two genes within the flagellar regulon, fliT and fliZ, have been shown to be involved in fine control of the class 2 operons (23). The fliT and fliZ genes belong to the fliDST and fliAZ operons, respectively, both of which are transcribed from both class 2 and class 3 promoters (12, 21, 46). FliT acts as an anti-FlhD 4 C 2 factor, which binds to the FlhD 4 C 2 complex through interaction with the FlhC subunit and inhibits its binding to the class 2 promoter, resulting in decreased expression of the class 2 operons (45). On the other hand, FliZ was shown to be a positive regulator of class 2 operons (23) and to participate in a positive-feedback loop that induces a kinetic switch in class 2 operon expression (38). Although class 2 operon expression is inhibited in vivo by the fliZ...

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“…Related experiments with Flp and Cre recombinase were interpreted to imply that this chirality may be shared by all λ-integrase family members. The simplest explanation for a chirality consistent with this proposal would be an out of plane rotation of the two loxP sites during recombination, resulting in a righthanded crossing of the sites (65,66). There are currently no structural models to support this idea because all tyrosine recombinase crystal structures of synaptic complexes and HJ intermediates feature core half-sites that are nearly co-planar.…”

Section: Alternative Models Of the Synaptic Complexmentioning

confidence: 97%

Cre Recombinase

Duyne

2015

Microbiol Spectr

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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. In addition to the numerous in vivo and in vitro applications that have been reported since Cre was first shown to function in yeast and mammalian cells nearly 30 years ago, the Cre-loxP 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. This review discusses advances in the Cre recombinase field that have occurred over the past 12 years since the publication of Mobile DNA II. The focus is on those recent contributions that have provided new mechanistic insights into the reaction. Also discussed are modifications of Cre and/or the loxP sequence that have led to improvements in genome engineering applications.

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DNA Topology and Geometry in Flp and Cre Recombination

Cited by 57 publications

References 52 publications

Synapsis of loxP Sites by Cre Recombinase

Synapsis of loxP Sites by Cre Recombinase

FliZ Acts as a Repressor of the ydiV Gene, Which Encodes an Anti-FlhD ₄ C ₂ Factor of the Flagellar Regulon in Salmonella enterica Serovar Typhimurium

Cre Recombinase

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DNA Topology and Geometry in Flp and Cre Recombination

Cited by 57 publications

References 52 publications

Synapsis of loxP Sites by Cre Recombinase

Synapsis of loxP Sites by Cre Recombinase

FliZ Acts as a Repressor of the ydiV Gene, Which Encodes an Anti-FlhD 4 C 2 Factor of the Flagellar Regulon in Salmonella enterica Serovar Typhimurium

Cre Recombinase

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Product

Resources

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FliZ Acts as a Repressor of the ydiV Gene, Which Encodes an Anti-FlhD ₄ C ₂ Factor of the Flagellar Regulon in Salmonella enterica Serovar Typhimurium