Interactions of the excision proteins of CTnDOT in the attR intasome

Excision of the conjugative transposon CTnDOT from the chromosome of Bacteroides spp. involves four CTnDOT-encoded proteins: IntDOT, Xis2c, Xis2d, and Exc along with a host factor. These proteins form excisive intasomes on the attR and attL sites which undergo synapsis and recombination to form the attDOT and attB sites. We recently developed an in vitro intramolecular excision reaction where the attL and attR sites are on the same plasmid. This reaction requires IntDOT, Xis2c, Xis2d, and is stimulated by Exc. We used this reaction to identify the binding sites of the IntDOT, Xis2c, and Xis2d. In this paper, we show that three of the six arm-type sites are absolutely required for excision. Furthermore, we also identified two binding sites for Xis2d and two possible binding sites for Xis2c on the attR site. We also showed that IntDOT interacts cooperatively with the Xis2c and Xis2d proteins on the attR site.