Homology-dependent interactions determine the order of strand exchange by IntDOT recombinase

The Bacteroides conjugative transposon CTnDOT encodes an integrase, IntDOT, which is a member of the tyrosine recombinase family. Other members of this group share a strict requirement for sequence identity within the region of strand exchange, called the overlap region. Tyrosine recombinases catalyze recombination by making an initial cleavage, strand exchange and ligation, followed by strand swapping isomerization requiring sequence identity in the overlap region, followed by the second cleavage, strand exchange and ligation. IntDOT is of particular interest because it has been shown to utilize a three-step mechanism: a sequence identity-dependent initial strand exchange that requires two base pairs of complementary DNA at the site of cleavage; a sequence identity-independent strand swapping isomerization, followed by a sequence identity-independent cleavage, strand exchange and ligation. In addition to the sequence identity requirement in the overlap region, Lambda Int interactions with arm-type sites dictate the order of strand exchange regardless of the orientation of the overlap region. Although IntDOT has an arm-binding domain, we show here that the location of sequence identity within the overlap region dictates where the initial cleavage takes place and that IntDOT can recombine substrates containing mismatches in the overlap region so long as a single base of sequence identity exists at the site of initial cleavage

CTnDOT integrase performs ordered homology-dependent and homology-independent strand exchanges

Although the integrase (IntDOT) of the Bacteroides conjugative transposon CTnDOT has been classified as a member of the tyrosine recombinase family, the reaction it catalyzes appears to differ in some features from reactions catalyzed by other tyrosine recombinases. We tested the ability of IntDOT to cleave and ligate activated attDOT substrates in the presence of mismatches. Unlike other tyrosine recombinases, the results revealed that IntDOT is able to perform ligation reactions even when all the bases within the crossover region are mispaired. We also show that there is a strong bias in the order of strand exchanges during integrative recombination. The top strands are exchanged first in reactions that appear to require 2 bp of homology between the partner sites adjacent to the sites of cleavage. The bottom strands are exchanged next in reactions that do not require homology between the partner sites. This mode of coordination of strand exchanges is unique among tyrosine recombinases