The organization of the outside end of transposon Tn5

Abstract

The end sequences of the IS50 insertion sequence are known as the outside end (OE) and inside end. These complex ends are related but nonidentical 19-bp sequences that serve as substrates for the activity of the Tn5 transposase. Besides providing the binding site of the transposase, the end sequences of a transposon contain additional types of information necessary for transposition. These additional properties include but are not limited to host protein interaction sites and sites that program synapsis and cleavage events. In order to delineate the properties of the IS50 ends, the base pairs involved in the transposase binding site have been defined. This has been approached through performing a variety of in vitro analyses: a hydroxyl radical missing-nucleoside interference experiment, a dimethyl sulfate interference experiment, and an examination of the relative binding affinities of single-site end substitutions. These approaches have led to the conclusion that the transposase binds to two nonsymmetrical regions of the OE, including positions 6 to 9 and 13 to 19. Proper binding occurs along one face of the helix, over two major and minor grooves, and appears to result in a significant bending of the DNA centered approximately 3 bp from the donor DNA-OE junction.

Tn 5 transposase mutants that alter DNA binding specificity 1 1Edited by G. Smith

“…Tn5 Tnp binds to OE and IE sequences in vitro (de la Cruz et al, 1993;Jilk et al, 1996). The role of speci®c base-pairs of the end sequences in transposition and in Tnp binding in particular has been studied through genetic analyses (Makris et al, 1988;Dodson & Berg, 1989;Tomcsanyi & Berg, 1989;Jilk et al, 1993) and chemical modi®-cation/inhibition analyses (Jilk et al, 1996).…”

DNA Sequence Bias During Tn5 Transposition

“…distorted in this region when bound to Tnp. 30,31 This suggests that the A/T enrichment observed during selection may be, in general, attributed to the ease of the distortion of weak base-pairs. In addition, it has been previously established that both nicked and abasic DNA substrates, which impart greater flexibility, significantly enhance the amount of DNA bound by Tnp.…”

Functional Characterization of Arginine 30, Lysine 40, and Arginine 62 in Tn5 Transposase

“…A possible explanation may be that a substantial DNA bend is found at or near the Ϫ1/ϩ1 cleavage site when the donor backbone is present in the transposase⅐DNA complex (22,23). Presumably, this bend is required for DNA binding when donor backbone is present.…”

“…q ϭ R40Q transposase complex; ϭ EKLP transposase complex. efficient binding of the transposon to Tn5 transposase is surprising, considering the large number of contact residues between the transposon end sequences and the transposase (21 The importance of individual specific interactions between the transposase and the transposon has been demonstrated by missing nucleoside experiments (7,8,22). Removal of individual nucleosides at positions ϩ4 to ϩ19 of the top nontransferred strand or ϩ5 to ϩ19 of the bottom transferred strand of the mosaic end sequences results in dramatic decreases in synaptic complex formation of EKLP transposase to the DNA (8).…”