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Chimeras of the Flp and Cre Recombinases: Tests of the Mode of Cleavage by Flp and Cre A

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Chimeras of the Flp and Cre Recombinases: Tests of the Mode of Cleavage by Flp and Cre A doi:10.1006/jmbi.2000.3967 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 302, 27±48 Chimeras of the Flp and Cre Recombinases: Tests of the Mode of Cleavage by Flp and Cre A. C. Shaikh and Paul D. Sadowski* Department of Molecular and The Flp and Cre recombinases are members of the integrase family of Medical Genetics, University of tyrosine recombinases. Each protein consists of a 13 kDa NH2-terminal Toronto, Toronto, M5S 1A8 domain and a larger COOH-terminal domain that contains the active site Canada of the enzyme. The COOH-terminal domain also contains the major determinants for the binding speci®city of the recombinase to its cognate DNA binding site. All family members cleave the DNA by the attach- ment of a conserved nucleophilic tyrosine residue to the 30-phosphate group at the sites of cleavage. In order to gain further insights into the determinants of the binding speci®city and modes of cleavage of Flp and Cre, we have made chimeric proteins in which we have fused the NH2-terminal domain of Flp to the COOH-terminal domain of Cre (``Fre'') and the NH2-terminal domain of Cre to the COOH-terminal domain of Flp (``Clp''). These chimeras have novel binding speci®cities in that they bind strongly to hybrid sites con- taining elements from both the Flp and Cre DNA targets but poorly to the native target sites. In this study we have taken advantage of the unique binding speci®ci- ties of Fre and Clp to examine the mode of cleavage by Cre, Flp, Fre and Clp. We ®nd that the COOH-terminal domain of the recombinases deter- mines their mode of cleavage. Thus Flp and Clp cleave in trans whereas Cre and Fre cleave in cis. These results agree with the studies of Flp and with the cocrystal structure of Cre bound to its DNA target site. They disagree with our previous ®ndings that Cre could carry out trans clea- vage. We discuss the variations in the experimental approaches in order to reconcile the different results. # 2000 Academic Press Keywords: Flp; Cre; site-speci®c recombination; DNA binding; chimeric *Corresponding author proteins Introduction et al., 1987; Lee et al., 1994; Qian et al., 1990), recom- bination takes place in a synaptic structure consist- The integrase family of conservative site-speci®c ing of two target sites each bound by two recombinases is comprised of over 100 members molecules of recombinase (Amin et al., 1991; Guo (Abremski & Hoess, 1992; Argos et al., 1986; et al., 1997, 1999; Hamilton & Abremski, 1984; Esposito & Scocca, 1997; Nunes-Duby et al., 1998). Hoess et al., 1990b). The crystal structure of the Cre These proteins all use a conserved catalytic tryo- synapse reveals an extensive series of cyclic NH2- sine residue to break and covalently attach to their terminal and COOH-terminal interactions that target sequences at speci®c phosphodiester bonds establish both ``cross-core'' interactions between (Craig, 1988; Evans et al., 1990; Gronostajski & the two protomers bound to the same lox site and Sadowski, 1985; Landy, 1989; Sadowski, 1993, ``synaptic''interactions between the Cre molecules 1995). The target sequences contain two inverted bound to the two lox sites in the synapse (Guo binding sites each of which binds a molecule of the et al., 1997). recombinase. Although cleavage can take place in Two of the most extensively characterized mem- a dimeric structure consisting of two recombinase bers of the integrase family are the Flp protein molecules bound to one target sequence (Andrews encoded by the 2 m plasmid of yeast (Sadowski, 1995) and the Cre protein of the bacteriophage P1 E-mail address of the corresponding author: (Hoess & Abremski, 1990). The ®rst step in the [email protected] recombination reaction is the site-speci®c binding 0022-2836/00/010027±22 $35.00/0 # 2000 Academic Press 28 Chimeras of the Flp and Cre Recombinases of the recombinase to its cognate recognition site, bond to be cleaved activates that bond to receive the FRT site for the Flp protein and the lox site for the catalytic tyrosine from another Flp protomer the Cre protein (Figure 1(a) and (b)). Although the bound to the target FRT sequence. While initial sequences of the sites differ, they share an identical experiments on the l integrase were compatible organization consisting of two inverted 13 bp with a trans mode of cleavage (Han et al., 1993), sequences (``symmetry elements'') surrounding an subsequent experiments using Holliday junctions 8 bp core region (Hoess et al., 1984, 1986; Lee & gave unequivocal evidence of cis cleavage (Nunes- Saito, 1998; Vetter et al., 1983). Duby et al., 1994). The XerC/XerD recombinase Early gel mobility shift and footprinting exper- also gave clear evidence for cis cleavage iments established the 13 bp symmetry elements as (Arciszewska & Sherratt, 1995). Our initial comple- the recognition elements for Flp and Cre (Andrews mentation experiments also indicated that the Cre et al., 1987; Mack et al., 1992). Hoess et al. (1990a) recombinase could cleave in trans (Shaikh & showed that the NH2-terminal domain of Cre pro- Sadowski, 1997). However, the cocrystal structure tected the core region of the lox site whereas the of Cre that was covalently attached to its DNA tar- COOH-terminal domain bound to the symmetry get showed that the cleavage had occurred in cis element. Missing contact probing of a single sym- (Guo et al., 1997). metry element showed that the core-proximal 4 bp The studies showing trans cleavage by Flp and of the symmetry were important for the binding of Cre made use of complementation experiments in intact Cre but not Cre25, the COOH-terminal which a given molecule of recombinase was posi- domain (Hoess et al., 1990a). The core-distal 9 bp tioned upon a full or half-target site (Chen et al., were needed for binding of both intact Cre and 1992; Shaikh & Sadowski, 1997). Trans cleavage Cre25 (Shaikh, 1997). Crosslinking studies using was demonstrated when one molecule of recombi- the NH2 and COOH-terminal domains of Flp nase donated its catalytic tyrosine to another showed a similar bipartite structure of the FRT recombinase protomer that was unable to cleave symmetry element. The core-proximal 4 bp were due to a mutation and was bound to a different important for the binding of the NH2-terminal site. domain, P13 and the COOH-terminal domain, P32 Such complementation experiments are subject bound to the core-distal 9 bp (Panigrahi & to the possible artifact that trans cleavage occurs Sadowski, 1994). because the design of the experiment precludes cis In order to gain further insight into the mechan- cleavage. The experiments with l integrase and ism of binding and cleavage by the Flp and Cre Holliday junctions gave unequivocal results recombinases, we have constructed chimeras because they made use of two integrases (l and between the two recombinases. The Fre protein HK022) of different binding speci®cities and the contains the NH2-terminal 13 kDa of Flp fused to authors were thereby able to position the proteins the COOH-terminal 25 kDa of Cre. The Clp protein at known sites in the Holliday substrate (Nunes- contains the NH2-terminal 13 kDa of Cre fused to Duby et al., 1994). Likewise the XerC and XerD the 32 kDa COOH-terminal domain of Flp. Each of proteins have different binding speci®cities and the chimeras binds site-speci®cally to hybrid sites therefore one can be certain of their position composed of sequence elements from the target during the cleavage experiment (Arciszewska & site of Flp and Cre, the FRT and lox sites. These Sherratt, 1995). novel binding speci®cities enabled us to examine In the present study we have made use of the the mode of cleavage by Flp, Cre, Fre and Clp. chimeric recombinases Clp and Fre to re-examine The members of the integrase family of recombi- the mode of cleavage by Cre and Flp. We exploited nases all employ a conserved nucleophilic tyrosine the altered binding speci®cities of the Clp and Fre that breaks the scissile phosphodiester bond and proteins to position them on hybrid binding covalently attaches the protein to the 30-phosphoryl elements called lrt and fox. We isolated heterodi- end at the site of the break (Argos et al., 1986; meric complexes containing one molecule of each Esposito & Scocca, 1997; Nunes-Duby et al., 1998). recombinase and assayed for covalent attachment However the family members display a diversity of the recombinases to the target site. We ®nd that of mechanisms of cleavage (Jayaram, 1997; the Cre and Fre proteins cleave in cis whereas the Jayaram & Lee, 1995). Cis cleavage means that the Flp and Clp proteins cleave in trans. We conclude molecule of the recombinase that donates the cata- that the COOH termini of the Cre and Flp proteins lytic tyrosine is bound immediately adjacent to the determine their mode of cleavage. scissile bond. Trans cleavage implies that the recombinase molecule that donates the tyrosine is not bound next to the scissile bond but resides else- Results where in the synaptic structure. Production of chimeric proteins Fre and Clp The Flp recombinase was the ®rst to be studied in this respect and all tests have showed that it Proteolysis of the Cre and Flp proteins had cleaves in trans (Chen et al., 1992; Lee et al., 1994, suggested a bidomainal structure for each protein 1999; Pan et al., 1993). This implies the active site (Hoess et al., 1990a; Pan & Sadowski, 1993; Pan of Flp is formed by contributions from two Flp et al., 1991). Furthermore, previous studies (Hoess protomers: the Flp molecule bound next to the et al., 1990a; Panigrahi & Sadowski, 1994; Shaikh, Chimeras of the Flp and Cre Recombinases 29 1997) as well as the recent cocrystal structure of distal 9 bp of the lox symmetry element is called Cre (Guo et al., 1997), suggested that each domain fox.
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