CTnDOT integrase (IntDOT) is a member of the tyrosine family of site-specific DNA recombinases. IntDOT is unusual in that it catalyzes recombination between nonidentical sequences. Previous mutational analyses centered on mutants with substitutions of conserved residues in the catalytic (CAT) domain or residues predicted by homology modeling to be close to DNA in the core-binding (CB) domain. That work suggested that a conserved active-site residue (Arg I) of the CAT domain is missing and that some residues in the CB domain are involved in catalysis. Here we used a genetic approach and constructed an Escherichia coli indicator strain to screen for random mutations in IntDOT that disrupt integrative recombination in vivo. Twenty-five IntDOT mutants were isolated and characterized for DNA binding, DNA cleavage, and DNA ligation activities. We found that mutants with substitutions in the amino-terminal (N) domain were catalytically active but defective in forming nucleoprotein complexes, suggesting that they have altered protein-protein interactions or altered interactions with DNA. Replacement of Ala-352 of the CAT domain disrupted DNA cleavage but not DNA ligation, suggesting that Ala-352 may be important for positioning the catalytic tyrosine (Tyr-381) during cleavage. Interestingly, our biochemical data and homology modeling of the CAT domain suggest that Arg-285 is the missing Arg I residue of IntDOT. The predicted position of Arg-285 shows it entering the active site from a position on the polypeptide backbone that is not utilized in other tyrosine recombinases. IntDOT may therefore employ a novel active-site architecture to catalyze recombination.Conjugative transposons (CTns) are mobile DNA segments that use conjugation and site-specific recombination to transfer a copy of their DNA from a donor to a recipient strain. CTnDOT was originally discovered in a strain of Bacteroides thetaiotaomicron that was capable of transferring resistance to tetracycline and erythromycin (4,35). Upon exposure to tetracycline, CTnDOT excises from the donor chromosome, copies its DNA by rolling-circle replication, and transfers its DNA to the recipient cell, where it circularizes and is integrated into the recipient chromosome by site-specific recombination. In the past 30 years, the frequency of tetracycline-resistant Bacteroides isolates has risen dramatically, to around 80% of isolates (35). Much of the spread of tetracycline resistance is due to the conjugative transposon CTnDOT and its close relatives (37).Previous work has shown that the integration and excision reactions require the CTnDOT-encoded integrase (IntDOT) and an uncharacterized Bacteroides host factor (8, 9, 30, 39). Analysis of the IntDOT amino acid sequence indicated that it was a member of the tyrosine recombinase family. It contains five of the six signature residues required for catalysis of the tyrosine recombination reactions (8,30,33). We previously constructed and characterized mutants containing alanine substitutions of residues in the catalytic (CAT...

CTnDOT encodes an integrase that is a member of the tyrosine recombinase family. The recombination reaction proceeds by sequential sets of genetic exchanges between the attDOT site in CTnDOT and an attB site in the chromosome. The exchanges are separated by 7 base pairs in each site. Unlike most tyrosine recombinases, IntDOT exchanges sites that contain different DNA sequences between the exchange sites to generate Holliday junctions (HJs) that contain mismatched bases. We demonstrate that IntDOT resolves synthetic HJs in vitro. Holliday junctions that contain identical sequences between the exchange sites are resolved into both substrates and products, while HJs that contain mismatches are resolved only to substrates. This result implies that resolution of HJs to products requires the formation of a higher-order nucleoprotein complex with natural sites containing IntDOT. We also found that proteins with substitutions of residues (V95, K94, and K96) in a putative alpha helix at the junction of the N and CB domains (coupler region) were defective in resolving HJs. Mutational analysis of charged residues in the coupler and the N terminus of the protein did not provide evidence for a charge interaction between the regions of the protein. V95 may participate in a hydrophobic interaction with another region of IntDOT.IntDOT is an integrase encoded by CTnDOT, a Bacteroides integrative and conjugative element (ICE). IntDOT is a member of the tyrosine family of recombinases, which includes lambda integrase, FLP, CRE, and the XerC and XerD recombinases. Lambda Int and IntDOT are called factor-assisted tyrosine recombinases, because they use accessory factors to regulate directionality of their recombination reactions. These recombinases contain three DNA binding domains: the Nterminal arm-binding domain (N), the core binding domain (CB), and the catalytic domain (CAT). These recombinases bind to two different types of DNA sites: the arm-type sites and core-type sites. For lambda Int, it has been shown that the N domain binds to the arm-type sites and functions in the directionality and regulation of catalysis. The CB and CAT domains of lambda Int bind to the core-type sites and perform catalysis (1,15,17).Site-specific recombination reactions catalyzed by most tyrosine recombinases occur by two sequential sets of strand exchanges. The sites of exchange border a region of DNA, called the overlap region, that is usually the same in both recombination sites. The nucleoprotein complexes that perform the recombination reactions contain four recombinase monomers bound to the four core-type sites of the partner DNA substrates. Two of the recombinase monomers are catalytically active, while the other two monomers are inactive. The first strand exchanges are carried out by the two active recombinase monomers to form a four-way branch structure Holliday junction (HJ) intermediate. In order to execute the second set of strand exchanges to form products, the HJ intermediate undergoes a conformational change that results in the activ...