Thiesen H J, Bach C
Basel Institute for Immunology, Switzerland.
FEBS Lett. 1991 May 20;283(1):23-6. doi: 10.1016/0014-5793(91)80545-e.
By substituting non conserved amino acids present in the postulated alpha-helical region of zinc finger domains, we demonstrated that Cys2/His2 type zinc finger domains could be targeted to new DNA binding sites. The putative alpha-helical region of the second SP1 zinc finger (RSDELQRH) was replaced by amino acids (KSSALISH) occurring in analogous zinc finger positions of human zinc finger protein Kox 29. The DNA binding specificity of the FPLC purified chimaeric protein (SP1-Kox 29) was determined by use of the target detection assay (TDA). Chimaeric protein SP1-Kox 29 was subjected to randomized oligonucleotides (GGG NNNN GGC) that were designed on the basis that each SP1 zinc finger interacts with 3-4 nucleotides concerning its cognate target site GGG GCGG GGC. By this analysis the DNA binding specificity of SP1-Kox 29 was shown to have switched from the cognate SP1 binding site to GGG GGTG GGC. Structure-function analysis of this type should facilitate the determination of DNA binding specificities for any individual zinc finger of interest.
通过替换锌指结构域假定的α螺旋区域中存在的非保守氨基酸，我们证明了Cys2/His2型锌指结构域可以靶向新的DNA结合位点。第二个SP1锌指（RSDELQRH）的假定α螺旋区域被人类锌指蛋白Kox 29类似锌指位置中出现的氨基酸（KSSALISH）所取代。使用靶标检测分析（TDA）确定了FPLC纯化的嵌合蛋白（SP1-Kox 29）的DNA结合特异性。嵌合蛋白SP1-Kox 29与随机寡核苷酸（GGG NNNN GGC）进行反应，这些寡核苷酸是基于每个SP1锌指与其同源靶位点GGG GCGG GGC的3至4个核苷酸相互作用而设计的。通过该分析表明，SP1-Kox 29的DNA结合特异性已从同源SP1结合位点转变为GGG GGTG GGC。这种类型的结构-功能分析应有助于确定任何感兴趣的单个锌指的DNA结合特异性。
