Transposon Tn7 inserts itself into the attTn7 target DNA sequence at the 3' end of the Escherichia coli glmS gene with high specificity and efficiency. This site in the E. coli genome displays amino acid conservation and nucleotide similarity with orthologous sequences in Archaebacteria and eukaryotes. Based on the high degree of nucleotide similarity, particularly with eukaryotes, we examined the interactions of a set of 20 bp duplex DNA sequences with the Tn7 protein TnsD. The protein was overexpressed in the IPTG-inducible vector pET14b-TnsD in E. coli BL21(DE3)-RIL-Codon-Plus, and purified by nickel chelation and ion exchange chromatography. Changes in the conformation of DNA duplexes upon interaction with TnsD were monitored by circular dichroism (CD) spectroscopy. TnsD binding to and dissociation from immobilized DNA duplexes were monitored by total internal reflectance (TIR). CD and TIR results were analyzed to calculate kon, koff, and KD values. The 20 bp DNA duplex corresponding to attTn7 at the 3' end of E. coli glmS displayed strong affinity for TnsD protein, with KD ~ 20 nM. Eukaryotic orthologs of attTn7 from yeast and mammalian GFPT1 displayed lower affinity, with KD ~ 500 nM. Mutant DNA sequences with a single central mismatch did not display any detectable interaction with TnsD. The physical studies validate our biological observation of Tn7 transposition into a plasmid containing the mammalian attTn7 ortholog sequence [Cleaver, S. H., and Wickstrom, E. (2000) Gene 254:37-44], and suggest that 1-2 amino acid substitutions in TnsD might be sufficient to permit binding to mammalian orthologs that is as strong as wild type TnsD binding to attTn7.