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Nucleic Acids Research, Vol 25, Issue 11 2153-2160, Copyright © 1997 by Oxford University Press

ARTICLES

DNA binding and phasing analyses of Tn5 transposase and a monomeric variant

D York and WS Reznikoff
Department of Biochemistry, 420 Henry Mall, University of Wisconsin- Madison, Madison, WI 53706, USA.

Both full-length Tn 5 transposase and a COOH-terminal truncated monomeric form of the protein,n369, have been shown to specifically bind end sequences at comparable affinities. In addition, both proteins distort the target sequence in a similar manner, as determined by a circular permutation assay. In this study,nEK54, a derivative ofn369 with a single amino acid substitution that significantly enhances binding activity, is used in further binding and bending studies along with full-length transposase. Phasing analysis has shown that distortion of the end sequences upon binding of full-length transposase and nEK54 protein is due in part to a protein-induced bend oriented towards the major groove. Because the center of transposase-induced bending maps to the extreme leftward end of the 19 bp consensus sequence, we examined the possibility that optimal protein binding requires additional upstream nucleotide contacts. Experiments presented here show that 9-10 nucleotides are needed upstream of +1 of the 19 bp sequence for efficient binding and this requirement can be met by either single-stranded or double-stranded DNA.
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