Determination of DNA binding specificities of mutated zinc finger domains

FEBS Letters

Bach, Christian¹  Thiesen, Hans-Jürgen¹ 

[1] Basel Institute for Immunology, Grenzacherstraße 487, Postfach CH-4005 Basel, Switzerland

关键词: Human transcription factor SPI;    Zinc finger protein domains;    DNA-protein interactions;    Zinc finger specific recognition code;   

DOI  :  10.1016/0014-5793(91)80545-E

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

By substituting non conserved amino acids present in the postulated α-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 α-helical region of the second SPI zinc finger (SLH) was replaced by amino acids (SLH) occurring in analogous zinc finger positions of human zinc finger protein Kox 29. The DNA binding specificity of the FPLC purified chimaeric protein (SPI-Kox 29) was determined by use of the target detection assay (TDA). Chimaeric protein SPI-Kox 29 wax subjected to randomized oligonucleotides (GGG NNNN GGC) that were designed on the basis that each SPI zinc finger interacts with 3–4 nucleotides concerning its cognate target site GGG GCGG GGC. By this analysis the DNA binding specificity of SPI-Kox 29 was shown to have switched from the cognate SPI 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.

【 授权许可】

Unknown