In vitro analysis of sequence requirements for the excision reaction of the Bacteroides conjugative transposon, CTnDOT.

CTnDOT, a Bacteroides conjugative transposon (CTn), initiates its transfer by excising to form a circular intermediate. This process has been shown to be complex, involving an unusual DNA intermediate with a short region of heterology and several CTn-encoded proteins. No information was available, however, about the sizes or sequence requirements of the att sites (attL and attR) at the ends of the integrated element where the processing occurs during excision. Using a newly developed in vitro competition excision assay, we have now localized attL to 153 bp and attR to 179 bp. Excision of CTnDOT involves staggered cuts that produce 5 bp chromosomal sequences at either end of the CTn. These 5 bp sequences (coupling sequences) form a region of heterology in the excised circular intermediate. Site-directed mutations that made the coupling sequences complementary and removed the region of heterology had no effect on excision. Thus, heterology is not essential. Mutagenesis of sequences adjacent to the coupling sequences revealed a 6 bp site in attR that was essential for excision. Mutating the analogous region in attL had little effect on excision. Regions within the attL site that appear to play a role in excision were found by introducing small insertions (phasing mutations) that could interfere with protein-protein or protein-DNA interactions. Similar insertion mutations in attR had no significant effect on excision. These results support the hypothesis that the CTnDOT excision reaction is asymmetrical with respect to likely protein binding sites and involves multiple protein-DNA interactions.