Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-01-28
2003-08-26
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C536S023100, C530S350000
Reexamination Certificate
active
06610512
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The field of this invention is zinc finger protein binding to target nucleotides. More particularly, the present invention pertains to amino acid residue sequences within the &agr;-helical domain of zinc fingers that specifically bind to target nucleotides of the formula 5′-(GNN)-3′.
BACKGROUND OF THE INVENTION
The paradigm that the primary mechanism for governing the expression of genes involves protein switches that bind DNA in a sequence specific manner was established in 1967 (Ptashne, M. (1967)
Nature
(London) 214, 3234). Diverse structural families of DNA binding proteins have been described. Despite a wealth of structural diversity, the Cys
2
-His
2
zinc finger motif constitutes the most frequently utilized nucleic acid binding motif in eukaryotes. This observation is as true for yeast as it is for man. The Cys
2
-His
2
zinc finger motif, identified first in the DNA and RNA binding transcription factor TFIIIA (Miller, J., McLachlan, A. D. & Klug, A. (1985)
Embo J
4, 1609-14), is perhaps the ideal structural scaffold on which a sequence specific protein might be constructed. A single zinc finger domain consists of approximately 30 amino acids with a simple &bgr;&bgr;&agr; fold stabilized by hydrophobic interactions and the chelation of a single zinc ion (Miller, J., McLachlan, A. D. & Klug, A. (1985)
Embo J
4, 1609-14, Lee, M. S., Gippert, G. P., Soman, K. V., Case, D. A. & Wright, P. E. (1989)
Science
245, 635-7). Presentation of the &agr;-helix of this domain into the major groove of DNA allows for sequence specific base contacts. Each zinc finger domain typically recognizes three base pairs of DNA (Pavletich, N. P. & Pabo, C. O. (1991)
Science
(Washington, D.C., 1883-) 252, 809-17, Elrod-Erickson, M., Rould, M. A., Nekludova, L. & Pabo, C. O. (1996)
Structure
(London) 4, 1171-1180, Elrod-Erickson, M., Benson, T. E. & Pabo, C. O. (1998)
Structure
(London) 6, 451-464, Kim, C. A. & Berg, J. M. (1996)
Nature Structural Biology
3, 940-945), though variation in helical presentation can allow for recognition of a more extended site (Pavletich, N. P. & Pabo, C. O. (1993)
Science
(Washington, D.C., 1883-) 261, 1701-7, Houbaviy, H. B., Usheva, A., Shenk, T. & Burley, S. K. (1996)
Proc Natl Acad Sci USA
93, 13577-82, Fairall, L., Schwabe, J. W. R., Chapman, L., Finch, J. T. & Rhodes, D. (1993)
Nature
(London) 366, 483-7, Wuttke, D. S., Foster, M. P., Case, D. A., Gottesfeld, J. M. & Wright, P. E. (1997)
J. Mol. Biol
. 273, 183-206). In contrast to most transcription factors that rely on dimerization of protein domains for extending protein-DNA contacts to longer DNA sequences or addresses, simple covalent tandem repeats of the zinc finger domain allow for the recognition of longer asymmetric sequences of DNA by this motif.
We have recently described polydactyl zinc finger proteins that contain 6 zinc finger domains and bind 18 base pairs of contiguous DNA sequence (Liu, Q., Segal, D. J., Ghiara, J. B. & Barbas III, C. F. (1997)
PNAS
94, 5525-5530). Recognition of 18 bps of DNA is sufficient to describe a unique DNA address within all known genomes, a requirement for using polydactyl proteins as highly specific gene switches. Indeed, control of both gene activation and repression has been shown using these polydactyl proteins in a model system (Liu, Q., Segal, D. J., Ghiara, J. B. & Barbas III, C. F. (1997)
PNAS
94, 5525-5530).
Since each zinc finger domain typically binds three base pairs of sequence, a complete recognition alphabet requires the characterization of 64 domains. Existing information which could guide the construction of these domains has come from three types of studies: structure determination (Pavietich, N. P. & Pabo, C. O. (1991)
Science
(Washington, D.C., 1883-) 252, 809-17, Elrod-Erickson, M., Rould, M. A., Nekludova, L. & Pabo, C. O. (1996)
Structure
(London) 4, 1171-1180, Elrod-Erickson, M., Benson, T. E. & Pabo, C. O. (1998)
Structure
(London) 6, 451-464, Kim, C. A. & Berg, J. M. (1996)
Nature Structural Biology
3, 940-945, Pavletich, N. P. & Pabo, C. O. (1993)
Science
(Washington, D.C., 1883-) 261, 1701-7, Houbaviy, H. B., Usheva, A., Shenk, T. & Burley, S. K. (1996)
Proc Natl Acad Sci USA
93, 13577-82, Fairall, L., Schwabe, J. W. R., Chapman, L., Finch, J. T. & Rhodes, D. (1993)
Nature
(London) 366, 483-7., 11, Wuttke, D. S., Foster, M. P., Case, D. A., Gottesfeld, J. M. & Wright, P. E. (1997)
J. Mol. Biol
. 273, 183-206., Nolte, R. T., Conlin, R. M., Harrison, S. C. & Brown, R. S. (1998)
Proc. Natl. Acad. Sci. U.S.A
. 95, 2938-2943, Narayan, V. A., Kriwacki, R. W. & Caradonna, J. P. (1997)
J. Biol. Chem
. 272, 7801-7809., site-directed mutagenesis (Isalan, M., Choo, Y. & Klug, A. (1997)
Proc. Natl. Acad. Sci. U.S.A
. 94, 5617-5621, Nardelli, J., Gibson, T. J., Vesque, C. & Charnay, P. (1991)
Nature
349, 175-178, Nardelli, J., Gibson, T. & Charnay, P. (1992)
Nucleic Acids Res
. 20, 4137.-44, Taylor, W. E., Suruki, H. K., Lin, A. H. T., Naraghi-Arani, P., Igarashi, R. Y., Younessian, M., Katkus, P. & Vo, N. V. (1995)
Biochemistry
34, 3222-3230, Desjarlais, J. R. & Berg, J. M. (1992)
Proteins: Struct., Funct., Genet
. 12, 101-4, Desjarlais, J. R. & Berg, J. M. (1992)
Proc Natl Acad Sci USA
89, 7345-9), and phage-display selections (Choo, Y. & Klug, A. (1994)
Proc Natl Acad Sci USA
91, 11163-7, Greisman, H. A. & Pabo, C. O. (1997)
Science
(Washington, D.C.) 275, 657-661.23, Rebar, E. J. & Pabo, C. O. (1994)
Science
(Washington, D.C., 1883-) 263, 671-3, Jamieson, A. C., Kim, S.-H. & Wells, J. A. (1994)
Biochemistry
33, 5689-5695, Jamieson, A. C., Wang, H. & Kim, S.-H. (1996)
PNAS
93, 12834-12839, Isalan, M., Klug, A. & Choo, Y. (1998)
Biochemistry
37, 12026-33, Wu, H., Yang, W.-P. & Barbas III, C. F. (1995)
PNAS
92, 344-348). All have contributed significantly to our understanding of zinc finger/DNA recognition, but each has its limitations. Structural studies have identified a diverse spectrum of protein/DNA interactions but do not explain if alternative interactions might be more optimal. Further, while interactions that allow for sequence specific recognition are observed, little information is provided on how alternate sequences are excluded from binding. These questions have been partially addressed by mutagenesis of existing proteins, but the data is always limited by the number of mutants that can be characterized. Phage-display and selection of randomized libraries overcomes certain numerical limitations, but providing the appropriate selective pressure to ensure that both specificity and affinity drive the selection is difficult. Experimental studies from several laboratories (Choo, Y. & Klug, A. (1994)
Proc Natl Acad Sci USA
91, 11163-7, Greisman, H. A. & Pabo, C. O. (1997)
Science
(Washington, D.C.) 275, 657-661, Rebar, E. J. & Pabo, C. O. (1994)
Science
(Washington, D.C., 1883-) 263, 671-3, Jamieson, A. C., Kim, S.-H. & Wells, J. A. (1994)
Biochemistry
33, 5689-5695.25, Jamieson, A. C., Wang, H. & Kim, S.-H. (1996)
PNAS
93, 12834-12839, Isalan, M., Klug, A. & Choo, Y. (1998)
Biochemistry
37, 12026-33), including our own (Wu, H., Yang, W.-P. & Barbas III, C. F. (1995)
PNAS
92, 344-348), have demonstrated that it is possible to design or select a few members of this recognition alphabet. However, the specificity and affinity of these domains for their target DNA was rarely investigated in a rigorous and systematic fashion in these early studies.
Since Jacob and Monod questioned the chemical nature of the repressor and proposed a scheme by which the synthesis of individual proteins within a cell might be provoked or repressed, specific experimental control of gene expression has been a tantalizing prospect (Jacob, F. & Monod, J. (1961)
J. Mol. Biol
. 3, 318-356). It is now well established that genomes are regulated at the level of transcription primarily through the action of proteins known as transcription factors that bind DNA in a sequence specific fashion. Often these protein factors act in a complex combinatorial ma
Carlson Karen Cochrane
Northrup Thomas E.
The Scripps Research Institute
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