DNA-binding activity and subunit interaction of the mariner transposase

Mos1 is a member of the mariner/Tc1 family of transposable elements origina lly identified in Drosophila mauritiana. It has 28 bp terminal inverted rep eats and like other elements of this type it transposes by a cut and paste mechanism, inserts at TA dinucleotides and codes for a transposase. This is the only protein required for transposition in vitro. We have investigated the DNA binding properties of Mos1 transposase and the role of transposase -transposase interactions in transposition. Purified transposase recognises the terminal inverted repeats of Mos1 due to a DNA-binding domain in the N -terminal 120 amino acids. This requires a putative helix-turn-helix motif between residues 88 and 108. Binding is preferentially to the right hand en d, which differs at four positions from the repeat at the left end. Cleavag e of Mos1 by transposase is also preferentially at the right hand end. Wild -type transposase monomers interact with each other in a yeast two-hybrid a ssay and we have used this to isolate mutations resulting in reduced intera ction. These mutations lie along the length of the protein, indicating that transposase-transposase interactions are not due to a single interaction d omain. One such mutation which retains both DNA-binding and catalytic activ ity has greatly reduced ability to excise Mos1 from plasmid DNA through coo rdinate cleavage of the two ends and transposition in vitro is lowered to a level 20-fold below that of the wild-type. This suggests that transposase- transposase interaction is required to form a synaptic complex necessary fo r coordinate cleavage at the ends of Mos1 during transposition. This mutant enzyme allows insertion at dinucleotides other than TA, including sequence s with GC base pairs. This is the first example of a mariner/Tc1 transposas e with altered target specificity.