IntDOT Interactions with Core Sites during Integrative Recombination

Integrative and conjugative elements (ICEs), formerly called conjugative transposons, have been implicated in the proliferation of antibiotic resistance genes. CTnDOT is an extensively studied ICE found in Bacteroides spp. In addition to carrying resistance genes to both erythromycin and tetracycline, CTnDOT carries a gene that encodes a tyrosine recombinase called IntDOT that catalyzes integration into and excision out of the bacterial host chromosome. CTnDOT integrates into one of several known attB sites in the bacterial chromosome that consists of a pair of inverted repeat core sites called B and B' in attB. The attDOT site contains the core sites and D and D'. These sites flank the overlap regions where strand exchanges occur. A notable feature of all known attB sites is the conservation of the B core site sequence, which is also found in the D core site of attDOT. In this study, we used a mutational analysis to establish the importance of this conserved sequence for integration and characterize the interaction of IntDOT with individual base pairs. We identified important T-A base pairs at position -5 in the B and D core sites and position -5 in the poorly conserved B' core site that are important for integrative recombination. Base analog studies suggest that IntDOT may make specific contacts with the A residues in the major groove at positions -5 and +5. IntDOT interaction with the A at position -5 in the B core site is required for the first strand exchange.