DNA-BINDING AND PHASING ANALYSES OF TN5 TRANSPOSASE AND A MONOMERIC VARIANT

Both full-length Tn5 transposase and a COOH-terminal truncated monomer ic form of the protein, Delta 369, have been shown to specifically bin d end sequences at comparable affinities. In addition, both proteins d istort the target sequence in a similar manner, as determined by a cir cular permutation assay. In this study, Delta EK54, a derivative of De lta 369 with a single amino acid substitution that significantly enhan ces binding activity, is used in further binding and bending studies a long with full-length transposase. Phasing analysis has shown that dis tortion of the end sequences upon binding of full-length transposase a nd Delta EK54 protein is due in part to a protein-induced bend oriente d towards the major groove. Because the center of transposase-induced bending maps to the extreme leftward end of the 19 bp consensus sequen ce, we examined the possibility that optimal protein binding requires additional upstream nucleotide contacts. Experiments presented here sh ow that 9-10 nucleotides are needed upstream of +1 of the 19 bp sequen ce for efficient binding and this requirement can be met by either sin gle-stranded or double-stranded DNA.