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Vanadate-based transition-state analog inhibitors of Cre-LoxP recombination

  • Author(s): Martin, SS
  • Wachi, S
  • Baldwin, EP
  • et al.
Abstract

Cre recombinase exchanges DNA strands at the LoxP recognition site via transphosphorylation reactions that involve pentacoordinate transition states. We demonstrate that meta-vanadate ion (VO3-) and appropriate DNA substrates assemble a transition-state analog-like complex in the Cre active site. Meta-vanadate inhibits recombination of LoxP-derived oligonucleotide substrates that contain a gap at either or both scissile phosphates, but does not inhibit reactions with intact LoxP. The 3′-hydroxyl group of the gapped substrate is required for inhibition, suggesting that vanadate is ligated by three oxo ligands. Assembly of the inhibited complex is slow (t1/2=19min at 4mM NaVO3) and requires Cre, substrates, and meta-vanadate. Holliday junction intermediates accumulated at lower meta-vanadate concentrations, suggesting that the second strand exchange is inhibited more readily than the first. The apparent KDfor meta-vanadate is 1.5-2mM and binding shows positive cooperativity. This methodology may have general application for mechanistic studies of recombinase/topoisomerase-mediated strand exchange reactions. © 2003 Elsevier Inc. All rights reserved.

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