Imanishi Miki, Nakamura Atsushi, Morisaki Tatsuya, Futaki Shiroh
Institute for Chemical Research, Kyoto University, Uji, Kyoto, Japan.
Biochem Biophys Res Commun. 2009 Sep 25;387(3):440-3. doi: 10.1016/j.bbrc.2009.07.059. Epub 2009 Jul 16.
One simple and widespread method to create engineered zinc fingers targeting the desired DNA sequences is to modularly assemble multiple finger modules pre-selected to recognize each DNA triplet. However, it has become known that a sufficient DNA binding affinity is not always obtained. In order to create successful zinc finger proteins, it is important to understand the context-dependent contribution of each finger module to the DNA binding ability of the assembled zinc finger proteins. Here, we have created finger-deletion mutants of zinc finger proteins and examined the DNA bindings of these zinc fingers to clarify the contributions of each finger module. Our results indicate that not only a positive cooperativity but also a context-dependent reduction in the DNA binding activity can be induced by assembling zinc finger modules.
一种简单且广泛应用的方法是模块化组装多个预先选择好的可识别每个DNA三联体的锌指模块，以此来构建靶向所需DNA序列的工程化锌指。然而，人们已经知道，并非总能获得足够的DNA结合亲和力。为了构建成功的锌指蛋白，了解每个锌指模块对组装后的锌指蛋白DNA结合能力的上下文依赖性贡献非常重要。在这里，我们构建了锌指蛋白的手指缺失突变体，并检测了这些锌指的DNA结合情况，以阐明每个手指模块的贡献。我们的结果表明，组装锌指模块不仅可以诱导正协同效应，还能诱导DNA结合活性的上下文依赖性降低。
