TN916 TARGET DNA-SEQUENCES BIND THE C-TERMINAL DOMAIN OF INTEGRASE PROTEIN WITH DIFFERENT AFFINITIES THAT CORRELATE WITH TRANSPOSON INSERTION FREQUENCY

The conjugative transposon Tn916 inserts with widely different frequencies into a variety of target sites with related nucleotide sequences, The binding of chimeric proteins, consisting of maltose-binding protein fused to Tn916 integrase, to three different target sequences for Tn916 was examined by DNase I protection experiments, The C-terminal DNA binding domain of the Tn916 integrase protein was shown to protect approximately 40 bp, spanning target sites in the orfA and car genes of the plasmid pIP501 and in the cylA gene of the plasmid pAD1, Competition binding assays showed that the affinities of the three target sites for Tn916 integrase varied over a greater than 3- but less than 10-fold range and that the cat target site bound integrase at a lower affinity than did the other two target sites, A PCR-based assay for transposition in Escherichia coli was developed to assess the frequency with which a defective minitransposon inserted into each target site, In these experiments, integrase provided in trans from a plasmid was the sole transposon encoded protein present, This assay detected transposition into the orfA and cylA target sites but not into the cat target site, Therefore, the frequency of transposon insertion into a particular target site correlated with the affinity of the target for the integrase protein, Sequences within the target fragments similar to known Tn916 insertion sites were not protected by integrase protein, Analysis of the electrophoretic behavior of circularly permuted sets of DNA fragments showed that all three target sites contained structural features consistent with the presence of a static bend, suggesting that these structural features in addition to the primary nucleotide sequence are necessary for integrase binding and, thus, target site activity.