Edwards Wesleigh F, Young Douglas D, Deiters Alexander
ACS Chem Biol. 2009 Jun 19;4(6):441-5. doi: 10.1021/cb900041s.
Cre recombinase catalyzes DNA exchange between two conserved lox recognition sites. The enzyme has extensive biological application, from basic cloning to engineering knock-out and knock-in organisms. Widespread use of Cre is due to its simplicity and effectiveness, but the enzyme and the recombination event remain difficult to control with high precision. To obtain such control we report the installation of a light-responsive o-nitrobenzyl caging group directly in the catalytic site of Cre, inhibiting its activity. Prior to irradiation, caged Cre is completely inactive, as demonstrated both in vitro and in mammalian cell culture. Exposure to non-damaging UVA light removes the caging group and restores recombinase activity. Tight spatio-temporal control over DNA recombination is thereby achieved.
Cre重组酶催化两个保守的lox识别位点之间的DNA交换。该酶具有广泛的生物学应用，从基础克隆到构建基因敲除和基因敲入生物体。Cre的广泛应用归因于其简单性和有效性，但该酶及其重组事件仍难以高精度控制。为了实现这种控制，我们报道了在Cre的催化位点直接安装一个光响应性邻硝基苄基笼蔽基团，从而抑制其活性。在照射之前，笼蔽的Cre完全无活性，这在体外和哺乳动物细胞培养中均得到证实。暴露于无损伤的紫外光A可去除笼蔽基团并恢复重组酶活性。从而实现了对DNA重组的严格时空控制。
