Importance of the conserved TG/CA dinucleotide termini in phage Mu transposition: similarities to transposable elements in the human genome

The dinucleotide TG/CA found at the termini of transposable phage Mu also occurs at the termini of a large class of transposable elements (TEs). In order to understand its importance, the activity of all 16 dinucleotide permutations of the termini was examined using a sensitive plasmid-based in vivo transposition assay. The reactivity of these substrates varied over several orders of magnitude in vivo, with substitutions at the T/A position being more severely impaired than those at the G/C position. The same general heirarchy of reactivity was observed in vitro using mutant oligonucleotide substrates. These experiments revealed that TG/CA was important not for the chemistry of strand transfer but for the stage of assembly of a stable transpososome. Given that DNA at the Mu-host junctions is melted/distorted concomitantly with transpososome assembly, we hypothesized that the terminal TG/CA dinucleotide has been selected primarily for its conformational flexibility. To test this hypothesis, we examined the activity of substrates carrying a hundred different pairs of mismatched termini. Consistent with the flexibility hypothesis, we found that mismatched substrates are extremely efficient at assembly. A wild-type T residue on the bottom strand is essential for stable assembly, but the identity of the dinucleotide on the top strand is irrelevant for transposition chemistry. In addition, we have found a new rule for suppression of terminal defects by MuB protein, as well as a role for metal ions in DNA opening at the termini. To extend the flexibility hypothesis to other TEs that perform DNA cleavage and strand transfer at precise DNA positions, we performed a statistical analysis of sequences found at the termini of precise TEs in the human genome. The results showed that LTR retrotransposons and DNA transposons encode the most flexible dinucleotide (TG/CA) and trinucleotide (CAG/CTG) most frequently at their termini, respectively. Combining results from this statistical analysis with previous findings in phage Mu transposition, we propose that a flexible terminal 2-3 base pair step is a core component of the machinery of precise TEs, and that molecular interactions at the +1 position influence a rate-limiting step.