Formation, characterization and partial purification of a Tn5 strand transfer complex

DNA transposition reactions typically involve a strand transfer step wherein the transposon ends are covalently joined by the transposase protein to a short target site. There is very little known about the transposase-DNA interactions that direct this process, and thus our overall understanding of the dynamics of DNA transposition reactions is limited. Tn5 presents an attractive system for defining such interactions because it has been possible to solve the structure of at least one Tn5 transposition intermediate: a transpososome formed with pre-cleaved ends. However, insertion specificity in the Tn5 system is low and this has hampered progress in generating target-containing transpososomes that are homogeneous in structure (i.e. where a single target site is engaged) and therefore suitable for biochemical and structural analysis. We have developed a system where the Tn5 transpososome integrates almost exclusively into a single target site within a short DNA fragment. The key to establishing this high degree of insertion specificity was to use a target DNA with tandem repeats of a previously characterized Tn5 insertion hotspot. The target DNA requirements to form this strand transfer complex are evaluated. In addition, we show that target DNAs missing single phosphate groups at specific positions are better substrates for strand transfer complex formation relative to the corresponding unmodified DNA fragments. Moreover, utilization of missing phosphate substrates can increase the degree of target site selecticin. A method for concentrating and partially purifying the Tn5 strand transfer complex is described. (c) 2006 Elsevier Ltd. All rights reserved.