Determination of DNA binding specificities of mutated zinc finger domains

Abstract

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.

Repression of transcriptional activity by heterologous KRAB domains present in zinc finger proteins

“…The chimeric KRAB domain between the A subdomain of ZNFI36 and the B subdomain of ZNF 10 was constructed by making use of PCR based site directed mutagenesis [29]. Two PCR reactions were run, one with the cDNA of ZNF136, the other with the cDNA of ZNF10.…”

Use of Multi Threaded Asynchronous DNA Sequence Pattern Searching Tool to Identifying Zinc-Finger-Nuclease Binding Sites on the Human Genome

“…Sp1 consists of a three zinc finger binding domain wherein we altered amino acids in its 2 nd finger in order to produce Sp1-mutants with changed binding site recognition (Thiesen & Bach, 1991). A similar strategy has been employed with phage libraries that comprise of up to 10 7 mutants of the middle finger of three finger biding domain of the mammalian zinc finger protein Zif268 (Klug, 2010).…”

“…Figure With site directed PCR mutagenesis (Thiesen & Bach, 1991), (Berg, 1988) and sequencing, Sp1 mutants CB1( Figure 2) and MR14 ( Figure 3) have been produced. Figure 2 and Figure 3 show the altered amino acids of CB1 and MR14 in double underline/italic/red.…”

“…However, zinc finger nucleases (ZNFs) are causing cytotoxicity (cell death) which is a serious setback for using them for clinical purposes [6], [7]. This is due to limited understanding of "even simple protein-DNA interactions [8] p. 2500", [9], despite rapid progress in the field [10] since the first mutated protein has been reported [11].…”

Evaluation of Novel Design Strategies for Developing Zinc Finger Nucleases Tools for Treating Human Diseases

“…In an effort to reduce complexity and to develop solutions in a timely fashion, it might be realistic to use a natural zinc finger binding domain and exchange amino acids in the alpha helical region of one of the fingers to change the domains' binding preferences [8, 13]. To test the feasibility of changing the rank-ordered binding preferences, the three-finger binding domain of SP1 is used to reduce complexity by focusing on exchanging amino acids in the alpha helical region of the 2nd finger.…”