Structure-Function Analysis of IntDOT

Abstract

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...

Resolution of Holliday Junction Recombination Intermediates by Wild-Type and Mutant IntDOT Proteins

“…coli in vivo integration and in vitro assays. In vivo integration and DNA binding and ligation assays were performed as described previously (8).…”

“…Recent in vitro experiments and homology modeling indicated that R285 might provide the function of the missing arginine (8,12). This arginine residue was predicted to project into the active site, where it could participate in catalysis.…”

“…IntDOT mutants deficient in integration, listed in Table 1, were characterized previously in DNA binding, cleavage, and ligation assays (8). We showed previously that IntDOT and E. coli integration host factor (IHF) form a gel shift complex with attDOT DNA.…”

“…We do not know the exact number of IntDOT monomers bound, but it is likely that the IHF bends the DNA, facilitating the binding of at least one IntDOT that binds simultaneously to a core-and arm-type site. The cleavage and ligation assays measure the ability of the wild-type or mutant IntDOT proteins to perform catalytic functions independent of strand exchange (8).…”

“…This study focuses on characteristics of HJ resolution by wild-type and mutant IntDOT proteins (8) and the roles of the N domain, the coupler region between the N and CB domains, and other residues of IntDOT in resolution of HJs. We showed that IntDOT resolves synthetic HJs in vitro.…”