Structural Studies of DNA- binding Proteins 
ognize B-DNA and fit into the major groove. Each 
finger makes its primary contacts with a 3 -base 
pair "subsite," and side chains near the amino- 
terminal end of the a-helix make the critical con- 
tacts with the bases. 
Since the zinc fingers are used in a modular 
fashion, they may be the ideal motif to use as we 
try designing novel DNA-binding proteins. How- 
ever, we need to determine the structure of addi- 
tional zinc finger-DNA complexes so that we can 
see how this motif is used in other proteins. (Do 
all zinc fingers dock against the DNA in the same 
way?) Nikola Pavletich has cloned the zinc finger 
regions from a number of other proteins and has 
recently solved the structure of a complex that 
contains the five zinc fingers from GLI, a protein 
that is amplified in a subset of human tumors. 
Finishing this structure and comparing it with the 
zif complex should provide a firmer basis for the 
design projects. 
Structural Studies of Other Protein-DNA 
Complexes 
It is important to obtain structural information 
about the other major DNA-binding motifs that 
occur in eukaryotic regulatory proteins. We are 
focusing on the helix-loop-helix proteins and the 
POU domain, which have very important roles in 
development. We also are working with the 
TFIID protein (because of its central role in tran- 
scription) and with the p53 protein, which is the 
most common site of mutations in human tumors. 
Because structural analysis often is limited by 
the ability to obtain suitable crystals, we are try- 
ing to improve methods for the cocrystallization 
of protein-DNA complexes. Our initial approach 
involved systematic changes in the length of the 
DNA site and required that the entire site be 
resynthesized for each experiment. We now have 
encouraging preliminary results with a linker co- 
crystallization scheme that combines the protein, 
the binding site, and a library of DNA linkers that 
can be used with any complex. This strategy may 
allow a dramatic increase in the number of co- 
crystallization conditions that can be tested. 
Design of Novel DNA-binding Proteins 
We are attempting to use the zinc finger struc- 
tures as a basis for designing novel DNA-binding 
proteins. Two major approaches are being tested: 
1 ) genetic strategies for selecting zinc finger pro- 
teins that recognize desired target sequences and 
2) strategies for computer-aided protein design. 
These programs, which can systematically con- 
sider a large number of sequences and conforma- 
tions, are being used in our attempts to design 
zinc finger proteins that will recognize novel 
binding sites. This combination of structural anal- 
ysis, computer-aided protein design, and genetic 
selection should provide a better understanding 
of protein-DNA recognition and allow rapid de- 
sign of zinc finger proteins that recognize novel 
target sites. 
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