Vanadate-based transition-state analog inhibitors of Cre-LoxP recombination.
Vanadate-based transition-state analog inhibitors of Cre-LoxP recombination.
复制标题
基于钒酸盐的 Cre-LoxP 重组过渡态类似物抑制剂。
DOI：
10.1016/s0006-291x(03)01437-2
复制
发表时间：
2003
影响因子：
3.1
通讯作者：
Baldwin,EnochP
中科院分区：
文献类型：
--
作者：
Martin,ShelleyS;Wachi,Shinichiro;Baldwin,EnochP
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.
