Vanadate-based transition-state analog inhibitors of Cre-LoxP recombination.
- Author(s): Martin, Shelley S
- Wachi, Shinichiro
- Baldwin, Enoch P
- et al.
Published Web Locationhttps://doi.org/10.1016/s0006-291x(03)01437-2
Cre recombinase exchanges DNA strands at the LoxP recognition site via transphosphorylation reactions that involve pentacoordinate transition states. We demonstrate that meta-vanadate ion (VO(3)(-)) 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)=19min at 4mM NaVO(3)) 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 1.5-2mM and binding shows positive cooperativity. This methodology may have general application for mechanistic studies of recombinase/topoisomerase-mediated strand exchange reactions.