Tn916 target DNA sequences bind the C-terminal domain of integrase protein with different affinities that correlate with transposon insertion frequency

Abstract

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 cat 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.

Behavior and Target Site Selection of Conjugative Transposon Tn 916 in Two Different Strains of Toxigenic Clostridium difficile

“…Furthermore, in this work, we have shown that there is an extended consensus sequence in strain R20291 compared to that of 630, suggesting that there are differences between the two strains that influence target site selection by Tn916. This could be local variation in DNA topology, as it has been shown that Tn916 is likely to preferentially insert into targets that have a static bend (19).…”

An in silico evaluation of Tn916 as a tool for generalized mutagenesis in Haemophilus influenzae Rd

“…The secondary structure of Int 6 may therefore result in an optimal interaction with integrase which would explain why Tn916 integration occurs here more frequently than at other target sites. The affinity with which a target site binds integrase and hence the frequency with which it experiences Tn92 6 insertion may therefore be dictated in part by its secondary structure (Lu & Churchwood, 1995).…”

“…This observation may explain why Int 6 acts as a 'hot-spot' for Tn916 insertion. (Lu & Churchwood, 1995) were identified. These sites were not used by Tn916 in subsequent transformation experiments, indicating that it could integrate into the Rd genome at sites which did not fit the consensus sequence.…”

“…A characteristic of Tn916 target sequences is that they are predicted to contain static bends centred at the site of transposon insertion. This DNA topology is thought to facilitate binding of the integrase encoded by Tn916 and thereby integration of the transposon into the host genome (Lu & Churchwood, 1995). No single base in the consensus integration sequence is conserved amongst all target sequences analysed to date and individual targets can differ from this common sequence at several positions whilst retaining the ability to interact with integrase.…”

“…Integration of Tn916 into host DNA has been reported to occur at target sequences with the consensus 5' TT/ATTTT(N),AAAAAA/TA, where (N), represents any 6-base sequence (Lu & Churchwood, 1995). This consensus was derived by examination of 24 target sites (compiled by Scott et a!., 1994) used by Tn916 in Streptococcus pyogenes, Listeria monocytogenes, Bacillus subtilis, Ent.…”