Cre recombinase exchanges DNA strands at the LoxP recognition site via transphosphorylation reactions that involve penta-coordinate 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 (t(1/2) = 19 min at 4 mM 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 K-D for meta-vanadate is 1.5-2 mM and binding shows positive cooperativity. This methodology may have general application for mechanistic studies of recombinase/topoisomerase-mediated strand exchange reactions. (C) 2003 Elsevier Inc. All rights reserved.