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Papers In Press, published online ahead of print May 4, 2001
Dept. of Molecular Biology, BCC 550, The Scripps Research Institute, La Jolla, CA 92037
Corresponding Author: carlos{at}scripps.edu
In previous studies we have developed Cys2 -His2 zinc finger domains that specifically recognized each of the sixteen 5’-GNN-3’ DNA target sequences and could be used to assemble six-finger proteins that bind 18 basepair DNA sequences (Beerli, R. R., Dreier, B. and Barbas, C. F., III (2000), Proc. Natl. Acad. Sci. U. S. A. 97, 1495-1500). Such proteins provide the basis for the construction of artificial transcription factors to study gene/function relationships in the post-genomic era. Central to the universal application of this approach is the development of zinc finger domains that specifically recognize each of the 64 possible DNA triplets. Here we describe the construction of a novel phage display library that enables the selection of zinc finger domains recognizing the 5’-ANN-3’ family of DNA sequences. Library selections provided domains that in most cases showed binding specificity for the 3 bp target site that they were selected to bind. These zinc finger domains were used to construct 6-finger proteins that specifically bound their 18 bp target site with affinities in the pM to low nM range. When fused to regulatory domains, these proteins containing various numbers of 5’-ANN-3’ domains were capable of specific transcriptional regulation of a reporter gene and the endogenous human erbB-2 and erbB-3 genes. These results suggest that modular DNA recognition by zinc finger domains is not limited to the 5’-GNN-3’ family of DNA sequences and can be extended to the 5’-ANN-3’ family. The domains characterized in this work provide for the rapid construction of artificial transcription factors, thereby greatly increasing the number of sequences and genes that can be targeted by DNA-binding proteins built from pre-defined zinc finger domains.
J. Biol. Chem, 10.1074/jbc.M102604200
Submitted on March 22, 2001
Revised on May 4, 2001
Accepted on May 4, 2001
Development of zinc finger domains for recognition of the 5'-ANN-3' family of DNA sequences and their use in the construction of artificial transcription factors
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