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Biomedical subjects

Y Choo

Publications and source records attributed to Y Choo.

At least 19 recordsLinked to original sources
PubMed2001

Exploring the DNA-binding specificities of zinc fingers with DNA microarrays.

A key step in the regulation of networks that control gene expression is the sequence-specific binding of transcription factors to their DNA recognition sites. A more complete understanding of these DNA-protein interactions will permit a more comprehensive and quantitative mapping of the regulatory pathways within cells, as well as a deeper understanding of the potential functions of individual genes regulated by newly identified DNA-binding sites. Here we describe a DNA microarray-based method to characterize sequence-specific DNA recognition by zinc-finger proteins. A phage display library, prepared by randomizing critical amino acid residues in the second of three fingers of the mouse Zif268 domain, provided a rich source of zinc-finger proteins with variant DNA-binding specificities. Microarrays containing all possible 3-bp binding sites for the variable zinc fingers permitted the quantitation of the binding site preferences of the entire library, pools of zinc fingers corresponding to different rounds of selection from this library, as well as individual Zif268 variants that were isolated from the library by using specific DNA sequences. The results demonstrate the feasibility of using DNA microarrays for genome-wide identification of putative transcription factor-binding sites.

Amino Acid Sequence↗
PubMed2001

Design of polyzinc finger peptides with structured linkers.

Zinc finger domains are perhaps the most versatile of all known DNA binding domains. By fusing up to six zinc finger modules, which normally recognize up to 18 bp of DNA, designer transcription factors can be produced to target unique sequences within large genomes. However, not all continuous DNA sequences make good zinc finger binding sites. To avoid having to target unfavorable DNA sequences, we designed multizinc finger peptides with linkers capable of spanning long stretches of nonbound DNA. Two three-finger domains were fused by using either transcription factor IIIA for the Xenopus 5S RNA gene (TFIIIA) finger 4 or a non-sequence-specific zinc finger as a "structured" linker. Our gel-shift results demonstrate that these peptides are able to bind with picomolar affinities to target sequences containing 0--10 bp of nonbound DNA. Furthermore, these peptides display greater sequence selectivity and bind with higher affinity than similar six-finger peptides containing long, flexible linkers. These peptides are likely to be of use in understanding the behavior of polydactyl proteins in nature and in the targeting of human, animal, or plant genomes for numerous applications. We also suggest that in certain polydactyl peptides an individual finger can "flip" out of the major groove to allow its neighbors to bind shorter, nontarget DNA sequences.

Animals↗
PubMed2001

Improved DNA binding specificity from polyzinc finger peptides by using strings of two-finger units.

Multizinc finger peptides are likely to reach increased prominence in the search for the "ideal" designer transcription factor for in vivo applications such as gene therapy. However, for these treatments to be effective and safe, the peptides must bind with high affinity and, more importantly, with great specificity. Our previous research has shown that zinc finger arrays can be made to bind 18 bp of DNA with picomolar affinity, but also has suggested that arrays of fingers also may bind tightly to related sequences. This work addresses the question of zinc finger DNA binding specificity. We show that by changing the way in which zinc finger arrays are constructed--by linking three two-finger domains rather than two three-finger units--far greater target specificity can be achieved through increased discrimination against mutated or closely related sequences. These new peptides have the added capability of being able to span two short gaps of unbound DNA, although still binding with picomolar affinity to their target sites. We believe that this new method of constructing zinc finger arrays will offer greater efficacy in the fields of gene therapy and in the production of transgenic organisms than previously reported zinc finger arrays.

Animals↗
PubMed2001

Selection of zinc fingers that bind single-stranded telomeric DNA in the G-quadruplex conformation.

There is considerable interest in molecules that bind to telomeric DNA sequences and G-quadruplexes with specificity. Such molecules would be useful to test hypotheses for telomere length regulation, and may have therapeutic potential. The versatility and modular nature of the zinc finger motif makes it an ideal candidate for engineering G-quadruplex-binding proteins. Phage display technology has previously been widely used to screen libraries of zinc fingers for binding to novel duplex DNA sequences. In this study, a three-finger library has been screened for clones that bind to an oligonucleotide containing the human telomeric repeat sequence folded in the G-quadruplex conformation. The selected clones show a strong amino acid consensus, suggesting analogous modes of binding. Binding was found to be both sequence dependent and structure specific. This is the first example of an engineered protein that binds to G-quadruplex DNA, and represents a new type of binding interaction for a zinc finger protein.

Amino Acid Sequence↗
PubMed2001

Monoamine oxidase B inhibitors from the fruits of Opuntia ficus-indica var. saboten.

Three varieties of methyl citrate and 1-methyl malate were isolated from the fruits of Opuntia ficus-indica var. saboten Makino through in vitro bioassay-guided isolation for the inhibition on monoamine oxidase(MAO). The IC50 values for MAO-B of 1-monomethyl citrate, 1,3-dimethyl citrate, trimethyl citrate and 1-methyl malate were 0.19, 0.23, 0.61 and 0.25 mM, respectively. However, on MAO-A, their inhibitions showed only marginal activity.

Citric Acid↗
PubMed2001

A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter.

DNA-binding domains with predetermined sequence specificity are engineered by selection of zinc finger modules using phage display, allowing the construction of customized transcription factors. Despite remarkable progress in this field, the available protein-engineering methods are deficient in many respects, thus hampering the applicability of the technique. Here we present a rapid and convenient method that can be used to design zinc finger proteins against a variety of DNA-binding sites. This is based on a pair of pre-made zinc finger phage-display libraries, which are used in parallel to select two DNA-binding domains each of which recognizes given 5 base pair sequences, and whose products are recombined to produce a single protein that recognizes a composite (9 base pair) site of predefined sequence. Engineering using this system can be completed in less than two weeks and yields proteins that bind sequence-specifically to DNA with Kd values in the nanomolar range. To illustrate the technique, we have selected seven different proteins to bind various regions of the human immunodeficiency virus 1 (HIV-1) promoter.

Amino Acid Sequence↗
PubMed2000

Engineered zinc finger proteins that respond to DNA modification by HaeIII and HhaI methyltransferase enzymes.

Zinc finger modules are capable of specifically interacting with DNA that contains 5-methylcytosine (5-mC) in place of cytosine, suggesting that zinc finger-DNA binding could be regulated by extrinsic methylation of DNA. Here, we have used phage display to engineer zinc finger proteins that detect and discriminate DNA methylation by the prokaryotic enzymes HaeIII and HhaI. In these systems, zinc finger-DNA complexes are induced by DNA modification using the appropriate enzyme, which can therefore act as a switch. To further develop the specificity of the switch, zinc finger discrimination between 5-mC and thymine in DNA sequences is demonstrated despite the presence of the characteristic major groove methyl group that is common to both bases. Specificity was achieved using a DNA-binding strategy involving synergy between adjacent zinc fingers. We propose that engineered zinc fingers that recognise particular DNA modifications, such as sequence-specific DNA methylation, could be integrated into artificial regulatory circuits for the control of gene expression and other biological processes.

Bacteriophages↗
PubMed2000

Advances in zinc finger engineering.

Recently developments have been made in engineering sequence-specific zinc finger DNA-binding proteins. Advances in this area will soon make it routine to target proteins to specific DNA sequences associated with any given gene. The primary interest is in the regulation of gene expression using customised transcription factors. However, modular catalytic domains are also being developed in order to engineer chimaeric proteins with customised restriction enzyme, methylase and integrase activity.

Animals↗
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↗