Structure of Aart, a Designed Six-finger Zinc Finger Peptide, Bound to DNA

Segal, David J.; Crotty, Justin W.; Bhakta, Mital S.; Barbas, Carlos F.; Horton, Nancy C.

doi:10.1016/j.jmb.2006.08.016

Cited by 85 publications

(63 citation statements)

References 53 publications

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“…Although it is well known that amino acid positions À1, 3, and 6 within the recognition helix directly contact the DNA triplet, positions À2, 1, and 5 are also described as playing a role in binding to the phosphate backbone. 34 Our study suggests that serine at position À2 and arginine at position 6 are very important in binding to the phosphate DNA backbone and 5 0 -nucleotide, respectively. However, the roles of demonstrated that a transcriptional repressor protein, namely KRAB-HLTR3, was able to achieve 100-fold repression of transcription from the HIV-1 promoter.…”

Section: Discussionmentioning

confidence: 67%

Designed zinc finger protein interacting with the HIV‐1 integrase recognition sequence at 2‐LTR‐circle junctions

et al. 2009

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Integration of HIV-1 cDNA into the host genome is a crucial step for viral propagation. Two nucleotides, cytosine and adenine (CA), conserved at the 3 0 end of the viral cDNA genome, are cleaved by the viral integrase (IN) enzyme. As IN plays a crucial role in the early stages of the HIV-1 life cycle, substrate blockage of IN is an attractive strategy for therapeutic interference. In this study, we used the 2-LTR-circle junctions of HIV-1 DNA as a model to design zinc finger protein (ZFP) targeting at the end terminal portion of HIV-1 LTR. A six-contiguous ZFP, namely 2LTRZFP was designed using zinc finger tools. The designed motif was expressed and purified from E. coli to determine its binding properties. Surface plasmon resonance (SPR) was used to determine the binding affinity of 2LTRZFP to its target DNA. The level of dissociation constant (K d ) was 12.0 nM. The competitive SPR confirmed that 2LTRZFP specifically interacted with its target DNA. The qualitative binding activity was subsequently determined by EMSA and demonstrated the aforementioned correlation. In addition, molecular modeling and binding energy analyses were carried out to provide structural insight into the binding of 2LTRZFP to the specific and nonspecific DNA target. It is suggested that hydrogen-bonding interactions play a key role in the DNA recognition mechanisms of the designed ZFP. Our study suggested an alternative HIV therapeutic strategy using ZFP interference of the HIV integration process.

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Section: Discussionmentioning

confidence: 67%

Designed zinc finger protein interacting with the HIV‐1 integrase recognition sequence at 2‐LTR‐circle junctions

et al. 2009

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“…GinC5 was generated by fusing a hyperactive catalytic domain of the Gin invertase to the C5 zinc finger protein. The composite structure shown here is derived from those of DNA-bound ␥␦ resolvase (12) and the Aart zinc finger protein (14).…”

Section: Figmentioning

confidence: 99%

“…These proteins are composed of a series of modular zinc finger domains that each bind specifically to 3 or 4 base pairs of DNA (14). Synthetic zinc finger DNA-binding proteins have been generated for many sequences, using several approaches (15)(16)(17).…”

mentioning

confidence: 99%

Synthesis of programmable integrases

Gordley

Gersbach

Barbas

2009

Proc. Natl. Acad. Sci. U.S.A.

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104

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Accurate modification of the 3 billion-base-pair human genome requires tools with exceptional sequence specificity. Here, we describe a general strategy for the design of enzymes that target a single site within the genome. We generated chimeric zinc finger recombinases with cooperative DNA-binding and catalytic specificities that integrate transgenes with >98% accuracy into the human genome. These modular recombinases can be reprogrammed: New combinations of zinc finger domains and serine recombinase catalytic domains generate novel enzymes with distinct substrate sequence specificities. Because of their accuracy and versatility, the recombinases/integrases reported in this work are suitable for a wide variety of applications in biological research, medicine, and biotechnology where accurate delivery of DNA is desired.recombinases ͉ zinc finger ͉ gene delivery ͉ gene targeting ͉ protein engineering T he postgenomic era of medicine will be defined by our ability to achieve biological control through genetic reprogramming. New tools are needed to accurately rewrite the genomic script and specifically alter genes, gene expression, and epigenetic state at any desired loci. To date, no enzyme-natural or synthetic-has been able to accurately modify only a single targeted site within the human genome (1). Scientists in biology, biotechnology, stem cell research, and gene therapy currently rely on naturally occurring enzymes to perform functions like DNA integration and excision. However, these enzymes recognize multiple sites within the human genome, often resulting in off-target DNA integration and chromosomal translocation (2-6). Our recent work with serine resolvases and invertases led us to hypothesize that we could use a modular approach that capitalizes on cooperative specificity to design synthetic enzymes that would uniquely recognize a single site within the 3 billion-base-pair human genome and allow us to deliver DNA specifically to this site (Fig. 1A) (7).In their native contexts, serine resolvases and invertases selectively recombine target sites within the same DNA molecule. This intramolecular specificity is assured by obligate assembly of large protein complexes, wherein accessory factors bound at neighboring sites impose topological and spatial constraints on the recombination reaction (8). Hyperactive mutants of several serine resolvases and invertases have been discovered that efficiently catalyze unrestricted recombination between minimal dimer-binding sites (Table S1) (9, 10). Furthermore, unlike other site-specific recombinases, serine resolvases and invertases are well suited to synthetic reengineering. These enzymes are modular in both structure and function, each comprised of a distinct catalytic domain flexibly tethered to a small helix-turnhelix DNA-binding domain (DBD). However, these DBDs are poorly suited for accurate genomic recombination (11) because the recognition motifs are short (4-6 bp) and degenerate (12, 13).In contrast, zinc finger DNA-binding proteins recognize target sites of v...

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“…Since the structure of Snail1 had not been resolved, we could not anticipate the solvent-exposed basic residues in the carboxyl half of the protein. However, given that zinc fingers are structurally well conserved, we generated a threedimensional model of this part of the protein using the synthetic six-finger zinc finger Aart polypeptide bound to DNA as a template (Segal et al, 2006) (Fig. 2B).…”

Section: Characterization Of the Snail1 Nlsmentioning

confidence: 99%

“…The template used was the six-finger zinc finger Aart polypeptide bound to DNA (PDB code 2I13) (Segal et al, 2006). Raw structures obtained from fitting were subjected to steepest descent energy minimization and checked for packing errors.…”

Section: Modellingmentioning

confidence: 99%

Characterization of Snail nuclear import pathways as representatives of C2H2 zinc finger transcription factors

Mingot

Vega

Maestro³

et al. 2009

Journal of Cell Science

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Snail proteins are C2H2 class zinc finger transcription factors involved in different processes during embryonic development, as well as in several adult pathologies including cancer and organ fibrosis. The expression of Snail transcription factors is tightly regulated at the transcriptional level and their activity is modulated by their subcellular localization. Given the importance of this gene family in physiology and pathology, it is essential to understand the mechanisms by which Snail proteins are imported into or exported out of the nucleus. Here we show that several importins mediate the nuclear import of the human Snail proteins and we identify a unique nuclear localization signal (NLS), recognized by all the importins, that has been conserved during the evolution of the Snail family. This NLS is characterized by the presence of basic residues at defined positions in at least three consecutive zinc fingers. Interestingly, the consensus residues for importin-binding are also involved in DNA binding, suggesting that importins could prevent non-specific binding of these transcription factors to cytoplasmic polyanions. Importantly, the identified basic residues are also conserved in other families of C2H2 transcription factors whose nuclear localization requires the zinc finger region.

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Structure of Aart, a Designed Six-finger Zinc Finger Peptide, Bound to DNA

Cited by 85 publications

References 53 publications

Designed zinc finger protein interacting with the HIV‐1 integrase recognition sequence at 2‐LTR‐circle junctions

Designed zinc finger protein interacting with the HIV‐1 integrase recognition sequence at 2‐LTR‐circle junctions

Synthesis of programmable integrases

Characterization of Snail nuclear import pathways as representatives of C2H2 zinc finger transcription factors

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