Roles of Exc Protein and DNA Homology in the CTnDOT Excision Reaction

Excision from the chromosome is the first step during the transfer of conjugative transposons (CTns) to a recipient. We previously showed that the excision of CTnDOT is more complex than the excision of lambdoid phages and CTns such as Tn916 . The excisionin vivo of CTnDOT utilizes four CTnDOT-encoded proteins, IntDOT, Xis2c, Xis2d, and Exc, and a host factor. We previously developed anin vitro excision reaction where the recombination sitesattL andattR were located on different plasmids. The reaction was inefficient and did not require Exc, suggesting that the reaction conditions did not mimicin vivo conditions. Here, we report the development of an intramolecular excision reaction where theattL andattR sites are located on the same DNA molecule. We found that Exc stimulates the reaction 3- to 5-fold. The efficiency of the excision reaction was also dependent on the distance between theattL andattR sites and on the sequences of the overlap regions between the sites of the strand exchanges. Substrates with identical overlap sequences recombined more efficiently than ones with heterologous overlap sequences. This was surprising, because the integration reaction is not sensitive to heterology in the overlap regions of theattDOT andattB sites.