Toward rules relating zinc finger protein sequences and DNA binding site preferences.

Zinc finger proteins of the Cys2-His2 type consist of tandem arrays of domains, where each domain appears to contact three adjacent base pairs of DNA through three key residues. We have designed and prepared a series of variants of the central zinc finger within the DNA binding domain of Sp1 by using information from an analysis of a large data base of zinc finger protein sequences. Through systematic variations at two of the three contact positions (underlined), relatively specific recognition of sequences of the form 5'-GGGGN(G or T)GGG-3' has been achieved. These results provide the basis for rules that may develop into a code that will allow the design of zinc finger proteins with preselected DNA site specificity.     

A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions

We have developed a bacterial "two-hybrid" system that readily allows selection from libraries larger than 10(8) in size. Our bacterial system may be used to study either protein-DNA or protein-protein interactions, and it offers a number of potentially significant advantages over existing yeast-based one-hybrid and two-hybrid methods. We tested our system by selecting zinc finger variants (from a large randomized library) that bind tightly and specifically to desired DNA target sites. Our method allows sequence-specific zinc fingers to be isolated in a single selection step, and thus it should be more rapid than phage display strategies that typically require multiple enrichment/amplification cycles. Given the large library sizes our bacterial-based selection system can handle, this method should provide a powerful tool for identifying and optimizing protein-DNA and protein-protein interactions.     

Two distinct classes of Ran-binding sites on the nucleoporin Nup-358.

Nup-358 is a giant nucleoporin located at the tips of the cytoplasmic fibrils of the nuclear pore complex (NPC). Its contains four RBH (RanBP1-homologous) domains and a zinc finger domain with eight zinc finger motifs. Using three recombinant fragments of Nup-358 that comprise two of the RBH domains and the zinc finger domain, we show that both RanGDP and RanGTP bind to Nup-358 in vitro. The RBH domains bound either RanGDP or RanGTP. Interestingly, the zinc finger domain was found to bind RanGDP exclusively. Zinc chelation by EDTA treatment abolished the binding of RanGDP to the zinc finger domain without affecting the binding of Ran to the RBH domain. Ultrastructural studies with RanGDP-conjugated colloidal gold in digitonin-permeabilized cells showed a large number of Ran-binding sites on the cytoplasmic fibrils of the NPC. Of those, only a portion that is closer to the central axis of the NPC was sensitive to RanBP1 competition, suggesting that most of the RBH domains of Nup-358 are situated closer to the central axis of the NPC than the zinc finger domain. Thus, the RBH and the zinc finger domains of Nup-358 were identified as two different classes of Ran-binding sites with distinct, ultrastructural locations at the NPC.     

Generation of monoclonal antibodies specifically directed against the proximal zinc finger of HIV type 1 NCp7

The HIV-1 NCp7 contains two spatially close zinc fingers, required for the production of infectious particles. To investigate in more detail the function of the zinc finger domain, monoclonal antibodies were generated with a cyclic analog of the NCp7 proximal zinc finger. This analog was shown to bind zinc ions and to preserve the highly folded structure of the native peptide (Dong C-Z et al.: J Am Chem Soc 1995;117:2726-2731). We report here two monoclonal antibodies (2B10 and 4D3), which are the first monoclonal antibodies directed against CCHC NCp7 zinc fingers. Dot-blot experiments revealed that a few nanograms of synthetic NCp7 can be detected on a nitrocellulose membrane. Whereas 2B10 appears specific for an epitope located in sequence 19-27 of NCp7, 4D3 appears to be structurally specific. Immunocomplex affinities were evaluated, using BIAcore technology, to be up to 1 and 10 nM, respectively, for 2B10 and 4D3 in 100 mM NaCl. These antibodies were able to recognize NCp7 in the Gag polyprotein precursor and were shown to immunoprecipitate NCp7 from a cell supernatant. Moreover, NCp7-Vpr interaction mediated by the zinc fingers is inhibited by 2B10, emphasizing the role of these domains in the protein-protein complex. These results indicate that 2B10 and 4D3 behave as useful tools for studying both NC protein functions during the course of virion morphogenesis and the role played by its zinc finger domain at various steps in the retroviral life cycle.     

Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5'-GNN-3' DNA target sequences

We have taken a comprehensive approach to the generation of novel DNA binding zinc finger domains of defined specificity. Herein we describe the generation and characterization of a family of zinc finger domains developed for the recognition of each of the 16 possible 3-bp DNA binding sites having the sequence 5'-GNN-3'. Phage display libraries of zinc finger proteins were created and selected under conditions that favor enrichment of sequence-specific proteins. Zinc finger domains recognizing a number of sequences required refinement by site-directed mutagenesis that was guided by both phage selection data and structural information. In many cases, residues not expected to make base-specific contacts had effects on specificity. A number of these domains demonstrate exquisite specificity and discriminate between sequences that differ by a single base with >100-fold loss in affinity. We conclude that the three helical positions -1, 3, and 6 of a zinc finger domain are insufficient to allow for the fine specificity of the DNA binding domain to be predicted. These domains are functionally modular and may be recombined with one another to create polydactyl proteins capable of binding 18-bp sequences with subnanomolar affinity. The family of zinc finger domains described here is sufficient for the construction of 17 million novel proteins that bind the 5'-(GNN)6-3' family of DNA sequences. These materials and methods should allow for the rapid construction of novel gene switches and provide the basis for a universal system for gene control.     

Zinc finger as distance determinant in the flexible linker of intron endonuclease I-TevI

I-TevI, the phage T4 td intron-encoded endonuclease, recognizes a lengthy DNA target and initiates intron mobility by introducing a double-strand break in the homing site. The enzyme uses both sequence and distance determinants to cleave the DNA 23-25 bp upstream of the intron insertion site. I-TevI consists of an N-terminal catalytic domain and a C-terminal DNA-binding domain separated by a long, flexible linker. The DNA-binding domain consists of three subdomains: a zinc finger, a minor-groove binding alpha-helix, and a helix-turn-helix. In this study, a mutational analysis was undertaken to assess the roles of these subdomains in substrate binding and cleavage. Surprisingly, the zinc finger is not required for DNA binding or catalysis. Rather, the zinc finger is a component of the linker and directs the catalytic domain to cleave the homing site at a fixed distance from the intron insertion site. When the cleavage site (CS) is shifted outside a given range, wild-type I-TevI defaults to the fixed distance, whereas zinc-finger mutants have lost the distance determinant and search out the displaced cleavage sequences. Although counterintuitive, a protein containing a 19-aa deletion of the zinc finger can extend further than can wild-type I-TevI to cleave a distant CS sequence, and a Cys-to-Ala mutant of the ligands for zinc, nominally a longer protein, can retract to cleave at a closer CS sequence. Models are presented for the novel function of the zinc finger, as a molecular constraint, whereby intramolecular protein-protein interactions position the catalytic domain by "catalytic clamp" and/or "linker-organizer" mechanisms.     

RAG-1 interacts with the repeated amino acid motif of the human homologue of the yeast protein SRP1.

Genes for immunoglobulins and T-cell receptor are generated by a process known as V(D)J recombination. This process is highly regulated and mediated by the recombination activating proteins RAG-1 and RAG-2. By the use of the two-hybrid protein interaction system, we isolated a human protein that specifically interacts with RAG-1. This protein is the human homologue of the yeast SRP1 (suppressor of a temperature-sensitive RNA polymerase I mutation). The SRP1-1 mutation is an allele-specific dominant suppressor of a temperature-sensitive mutation in the zinc binding domain of the 190-kDa subunit of Saccharomyces cerevisiae RNA polymerase I. The human SRP cDNA clone was used to screen a mouse cDNA library. We obtained a 3.9-kbp cDNA clone encoding the mouse SRP1. The open reading frame of this cDNA encodes a 538-amino acid protein with eight degenerate repeats of 40-45 amino acids each. The mouse and human SRP1 are 98% identical, while the mouse and yeast SRP1 have 48% identity. After cotransfection of the genes encoding RAG-1 and human SRP1 into 293T cells, a stable complex was evident. Deletion analysis indicated that the region of the SRP1 protein interacting with RAG-1 involved four repeats. The domain of RAG-1 that associates with SRP1 mapped N-terminal to the zinc finger domain. Because this region of RAG-1 is not required for recombination and SRP1 appears to be bound to the nuclear envelope, we suggest that this interaction helps to localize RAG-1.