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J. Biol. Chem., Vol. 276, Issue 31, 29466-29478, August 3, 2001
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,
From The Skaggs Institute for Chemical Biology and the Department
of Molecular Biology, The Scripps Research Institute,
La Jolla, California 92037
In previous studies we have developed
Cys2-His2 zinc finger domains that
specifically recognized each of the 16 5'-GNN-3' DNA target sequences
and could be used to assemble six-finger proteins that bind 18-base
pair 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-base pair target site that they
were selected to bind. These zinc finger domains were used to construct
6-finger proteins that specifically bound their 18-base pair 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.
Recipient of a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft.
§
Current address: Cytos Biotechnology AG, Wagisstrasse 21, 8952 Zurich-Schlieren, Switzerland.
¶
To whom correspondence should be addressed: The Scripps
Research Institute, BCC-515, North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-9098; Fax: 858-784-2583; E-mail:
carlos@scripps.edu.
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