Structure-Function Analysis of IntDOT

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) domain that are conserved among tyrosine recombinases. The results supported the inclusion of IntDOT within the tyrosine family of recombinases. However, the catalytic core seemed to have an organization somewhat different from those of other tyrosine recombinases (30). In addition, we used a homology modeling method to identify residues in the core-binding (CB) domain that are predicted to be near the DNA (29). The results of alanine substitutions of several residues indicated that some residues in the CB domain are likely involved in catalysis.The information-directed mutagenesis approaches that we used previously with IntDOT are useful for producing amino acid substitutions at positions predicted to be important for protein function on the basis of methods such as sequence analysis or homology modeling. Because IntDOT has an arm-binding (N) domain in the N terminus of the protein about which relatively little is known, and because the CAT domain appeared to have an unusual structure not found in other family members, the in vitro approach is limited in its ability to produce useful substitution mutations affecting the functions of these domains. In order to complement our earlier work, we chose to use a structure-function approach similar to one used previously with λ Int (21). The strategy we used was to isolate substitution mutants of IntDOT generated by random mutagenesis using an in vivo screen for recombination activity. This approach produced amino acid substitutions in all three of the domains of IntDOT. Analysis of the mutants has uncovered novel amino acid substitutions that cause defects in different steps in the recombination pathway, such as DNA binding, DNA cleavage, and DNA ligation.