Selection and Evolution of Novel DNA-Binding Proteins From Principles to Applications

Selection and Evolution of Novel DNA-Binding Proteins From Principles to Applications 

Transcription factors are modular proteins consisting typically of a DNA-binding domain that provides for localization of the protein to a specific DNA address, and an effector domain that directs the type of activity to take place at the site (Cowell, 1994 Ptashne, 1997). The wide range of effector activities available provide significant advantages over 

Impressive amino-acid conservation was observed for recognition of the same nucleotide in different targets and the selected residues in many cases made good chemical sense (Fig. 10). For example, Asn in position 3 (Asn3) was virtually always selected to recognize adenine in the middle position, whether in the context of GAG, GAA, GAT, or GAC. Gln-1 and Arg-1 were always selected to recognize adenine or 

Specific delivery of a DNA-binding protein to a single site within the 3.5 billion bp human genome requires an address of at least 16 bp. 

The resulting six-finger proteins have been shown to bind their 18-bp DNA targets with subnanomolar affinity (Beerli et al, 1998 Beerli et al, 2000 Liu et al, 1997). Mutating either half of the target site resulted in a 100-fold loss in affinity, and mutating only 3-bp produced a thirty- 

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