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Biomedical subjects

Y Choo

Publications and source records attributed to Y Choo.

13 recordsLinked to original sources
PubMed1998

Comprehensive DNA recognition through concerted interactions from adjacent zinc fingers.

Zinc fingers are small DNA-binding modules noted for their occurrence in a large number of eukaryotic transcription factors, and their use in protein engineering. Although it was expected that zinc fingers can bind to a wide diversity of DNA sequences, previous studies using model zinc finger domains from Zif268 (and Sp1) have revealed a potential limitation to the DNA-binding specificity. For example, phage display selection of individual zinc fingers to recognize trinucleotide DNA subsites returned fingers that bound specifically only to triplets of the form GNN, i.e., triplets with guanine at the 5' end. Following our recently reported work [Isalan, M., Choo, Y., and Klug, A. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 5617-5621], we now show that this limitation can be overcome by the concerted randomization of certain amino acid positions in adjacent zinc fingers that specify overlapping DNA subsites. This illustrates an important mechanism underlying DNA recognition by arrays of zinc fingers, and points the way to improved strategies for the design of highly specific zinc finger proteins that bind any given nucleotide sequence.

Amino Acid Sequence↗
PubMed1998

End effects in DNA recognition by zinc finger arrays.

The paradigmatic DNA binding domain from the transcription factor Zif268 contains three zinc finger modules in tandem repeat. When bound to their cognate DNA site the fingers read out the sequence of one DNA strand by making a linear series of successive base contacts. It is shown that the base-specific protein-DNA contacts made from the ends of the Zif268 three-finger array contribute less to the stability of the intermolecular complex than do structurally equivalent contacts from more central regions of the DNA binding domain. The effect is akin to the end fraying observed in duplex nucleic acid molecules.

Amino Acid Sequence↗
PubMed1998

All wrapped up.

How is it possible that nine small repeated 'zinc finger' units (each spanning just 3 or 4 base pairs) can protect the whole 50 base pair binding site of TFIIIA and why should such a periodic protein structure give rise to such an asymmetric footprint on DNA? The crystal structure of the first six fingers of TFIIIA bound to 31 base pairs of DNA explains everything: not all zinc fingers act alike.

Animals↗
PubMed1997

Promoter-specific activation of gene expression directed by bacteriophage-selected zinc fingers.

It has been shown that sequence-specific DNA-binding domains containing zinc fingers can be selected from libraries displayed on filamentous bacteriophage. The affinity and specificity of these peptides are well characterised in vitro, but few data are available to demonstrate specific DNA binding and discrimination between closely related DNA sequences in vivo. Transient transactivation assays were performed in mammalian cells, using expression plasmids which produce different amounts of a model transcription factor containing a phage-selected zinc finger DNA-binding domain, and reporter plasmids which carry systematic variations of the promoter sequence. When the intracellular concentration of the transcription factor was appropriate, activation of gene expression was absolutely dependent on a promoter having the same DNA sequence as that originally used to select the zinc finger domain by phage display. However, excessive intracellular concentrations of the transcription factor resulted in some less-specific DNA binding, leading to gene activation from similar promoters containing a maximum of two base changes. Thus, provided delivery is carefully controlled, highly specific control of gene expression in vivo can be achieved using artificial transcription factors containing phage-selected zinc finger DNA-binding domains.

Amino Acid Sequence↗
PubMed1997

Synergy between adjacent zinc fingers in sequence-specific DNA recognition.

Zif268-like zinc fingers are generally regarded as independent DNA-binding modules that each specify three base pairs in adjacent, but discrete, subsites. However, crystallographic evidence suggests that a contact also can occur from the second helical position of one finger to the subsite of the preceding finger. Here we show for the three-finger DNA-binding domain of the protein Zif268, and a panel of variants, that deleting the putative contact from finger 3 can affect the binding specificity for the 5' base in the adjoining triplet, which forms part of the binding site of finger 2. This finding demonstrates that Zif268-like zinc fingers can specify overlapping 4-bp subsites, and that sequence specificity at the boundary between subsites arises from synergy between adjacent fingers. This has important implications for the design and selection of zinc fingers with novel DNA binding specificities.

Amino Acid Sequence↗
PubMed1997

Physical basis of a protein-DNA recognition code.

Can a stereochemical recognition code explain sequence-specific protein-nucleic acid interactions? Whereas a code that is generally applicable to DNA-binding proteins of all known structural families is unattainable, the indications are that a code can describe at least some of the interactions of classical zinc fingers with DNA. The crystal structures of related zinc finger-DNA complexes reveal a remarkable mode of interaction that sets the framework for this code, and recent biochemical studies have elucidated the intermolecular contacts (contingent on this framework) that result in specificity.

DNA↗
PubMed1995

Designing DNA-binding proteins on the surface of filamentous phage.

The strategy of molecular evolution by phage display recently has been applied to the study of interactions between protein and DNA. This technology will imminently enable DNA-binding proteins to be made to measure. In the first instance, this will greatly advance our understanding of protein-DNA interactions, but in the long term, it is expected to yield powerful tools for use in medicine and research.

Animals↗
PubMed1994

In vivo repression by a site-specific DNA-binding protein designed against an oncogenic sequence.

A DNA-binding peptide comprising three zinc-fingers has been engineered to bind specifically to a unique nine-base-pair region of a BCR-ABL fusion oncogene in preference to the parent genomic sequences. Binding to the target oncogene in chromosomal DNA is possible in transformed cells in culture, and results in blockage of transcription. Consequently, murine cells rendered independent of growth factors by the action of the oncogene revert to factor dependence upon transient transfection with a vector expressing the peptide.

Amino Acid Sequence↗
PubMed1994

Toward a code for the interactions of zinc fingers with DNA: selection of randomized fingers displayed on phage.

We have used two selection techniques to study sequence-specific DNA recognition by the zinc finger, a small, modular DNA-binding minidomain. We have chosen zinc fingers because they bind as independent modules and so can be linked together in a peptide designed to bind a predetermined DNA site. In this paper, we describe how a library of zinc fingers displayed on the surface of bacteriophage enables selection of fingers capable of binding to given DNA triplets. The amino acid sequences of selected fingers which bind the same triplet are compared to examine how sequence-specific DNA recognition occurs. Our results can be rationalized in terms of coded interactions between zinc fingers and DNA, involving base contacts from a few alpha-helical positions. In the paper following this one, we describe a complementary technique which confirms the identity of amino acids capable of DNA sequence discrimination from these positions.

Amino Acid Sequence↗
PubMed1994

Selection of DNA binding sites for zinc fingers using rationally randomized DNA reveals coded interactions.

In the preceding paper [Choo, Y. & Klug, A. (1994) Proc. Natl. Acad. Sci. USA 91, 11163-11167], we showed how selections from a library of zinc fingers displayed on phage yielded fingers able to bind to a number of DNA triplets. Here, we describe a technique to deal efficiently with the converse problem--namely, the selection of a DNA binding site for a given zinc finger. This is done by screening against libraries of DNA triplet binding sites randomized in two positions but having one base fixed in the third position. The technique is applied here to determine the specificity of fingers previously selected by phage display. We find that some of these fingers are able to specify a unique base in each position of the cognate triplet. This is further illustrated by examples of fingers which can discriminate between closely related triplets as measured by their respective equilibrium dissociation constants. Comparing the amino acid sequences of fingers which specify a particular base in a triplet, we infer that in most instances, sequence-specific binding of zinc fingers to DNA can be achieved by using a small set of amino acid-nucleotide base contacts amenable to a code.

Amino Acid Sequence↗
PubMed1993

A role in DNA binding for the linker sequences of the first three zinc fingers of TFIIIA.

Zinc fingers of the TFIIIA type are connected by short linker sequences between the structural units. Structural investigations by 2D NMR in solution and by X-ray crystallographic analyses of complexes with DNA point to a passive role for the linkers. We have therefore investigated the influence of the linker sequence on DNA binding using as a model the first three fingers of the protein TFIIIA. Insertion of certain heterologous linkers abolishes binding, and replacement of individual amino acids can reduce binding by factors of up to twenty-four.

Amino Acid Sequence↗