Vanadate-based transition-state analog inhibitors of Cre-LoxP recombination.
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.
Martin
Shelley S
SS
Section of Molecular and Cellular Biology, University of California, Davis 95616, USA.
Wachi
Shinichiro
S
Baldwin
Enoch P
EP
D001483:Base Sequence; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D019426:Integrases / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D011995:Recombination, Genetic / Q000187:drug effects*; D014638:Vanadates / Q000737:chemistry / Q000494:pharmacology*; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism
D004791:Enzyme Inhibitors; D014764:Viral Proteins; D014638:Vanadates; C045073:Cre recombinase; D019426:Integrases