The Hairpin Binding Module

An area of ResT near the end of the N-terminal domain has sequence similarity to an important motif present in bacterial cut-and-paste transposases (see Fig. 2). Cut-and-paste transposases mobilize transposons like Tn5 or Tn10 via complete excision of the element from the chromosome by the introduction of double-strand breaks into the transposon ends and by their subsequent transfer to new DNA sites (Haniford 2002; Reznikoff 2003). Extensive biochemical and structural work has identified the "YREK motif", in conjunction with a constellation of active site residues typical of the RNaseH family of integrases/nucleases, as being responsible for transpo-son excision (Allingham et al. 2001; Ason and Reznikoff 2002; Davies et al. 2000; Lovell et al. 2002; Naumann and Reznikoff 2002). These transposases break four DNA strands utilizing only two active sites. This is accomplished by transition through a DNA hairpin intermediate (Kennedy et al. 1998, 2000) at each transposon end followed by hydrolytic opening of the hairpins (which are formed on the transposon side of the Tn/chromosome junctions). Creating the DNA distortion required for hairpin formation is the role of the YREK motif (Allingham et al. 2001; Ason and Reznikoff 2002; Davies et al. 2000; Rice and Baker 2001). The YREK motif has two separable components: a hydrophobic pocket that forms stacking interactions with the penultimate base of the transposon end which is flipped out of the double helix, contributing most of the distortion required for changing the trajectory of the DNA strands to form the hairpin, and the YREK residues that make stabilizing contacts with the distorted DNA about to be hairpinned.

Mutation of the corresponding hydrophobic pocket in ResT (P139A, W141A and the double mutant) leads to a severely compromised resolvase; mutation of the YKEK motif produces a cold-sensitive defect (Bankhead and Chaconas 2004). Importantly, mutants in the hairpin-binding module of ResT, as this region has come to be known, show a defect at the DNA cleavage step, unlike the transposase mutants that manifest their failure to make DNA hairpins as a strand transfer defect (Allingham et al. 2001; Bankhead and Chaconas 2004). A pre-cleavage distortion of the rTel DNA between the scissile phosphates by the hairpin-binding module is inferred from the ob servation that introduction of a heteroduplex into the central 2 bp of the rTel leads to a rescue of the cleavage defect of these mutants (DNA hairpinning then proceeds normally). The rationale is that the small region of heteroduplex around the symmetry axis of the rTel mimics the pre-cleavage action of this part of the protein.

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