An engineered split M.HhaI-zinc finger fusion lacks the intended methyltransferase specificity

The ability to site-specifically methylate DNA in vivo would have wide applicability to the study of basic biomedical problems as well as enable studies on the potential of site-specific DNA methylation as a therapeutic strategy for the treatment of diseases. Natural DNA methyltransferases lack the specificity required for these applications. Nomura and Barbas [W. Nomura, C.F. Barbas 3rd, In vivo site-specific DNA methylation with a designed sequence-enabled DNA methylase, J. Am. Chem. Soc. 129 (2007) 8676-8677] have reported that an engineered DNA methyltransferase comprised of fragments of M.HhaI methyltransferase and zinc finger proteins has very high specificity for the chosen target site. Our analysis of this engineered enzyme shows that the fusion protein methylates target and non-target sites with similar efficiency.     

Evolutionary analysis of a novel zinc ribbon in the N-terminal region of threonine synthase

Threonine synthase (TS) catalyzes the terminal reaction in the biosynthetic pathway of threonine and requires pyridoxal phosphate as a cofactor. TSs share a common catalytic domain with other fold type II PALP dependent enzymes. TSs are broadly grouped into two classes based on their sequence, quaternary structure, and enzyme regulation. We report the presence of a novel zinc ribbon domain in the N-terminal region preceding the catalytic core in TS. The zinc ribbon domain is present in TSs belonging to both classes. Our sequence analysis reveals that archaeal TSs possess all zinc chelating residues to bind a metal ion that are lacking in the structurally characterized homologs. Phylogenetic analysis suggests that TSs with an N-terminal zinc ribbon likely represents the ancestral state of the enzyme while TSs without a zinc ribbon must have diverged later in specific lineages. The zinc ribbon and its N- and C-terminal extensions are important for enzyme stability, activity and regulation. It is likely that the zinc ribbon domain is involved in higher order oligomerization or mediating interactions with other biomolecules leading to formation of larger metabolic complexes.     

Re-programming DNA-binding specificity in zinc finger proteins for targeting unique address in a genome

Recent studies provide a glimpse of future potential therapeutic applications of custom-designed zinc finger proteins in achieving highly specific genomic manipulation. Custom-design of zinc finger proteins with tailor-made specificity is currently limited by the availability of information on recognition helices for all possible DNA targets. However, recent advances suggest that a combination of design and selection method is best suited to identify custom zinc finger DNA-binding proteins for known genome target sites. Design of functionally self-contained zinc finger proteins can be achieved by (a) modular protein engineering and (b) computational prediction. Here, we explore the novel functionality obtained by engineered zinc finger proteins and the computational approaches for prediction of recognition helices of zinc finger proteins that can raise our ability to re-program zinc finger proteins with desired novel DNA-binding specificities.     

Pb-207 NMR spectroscopy reveals that Pb(II) coordinates with glutathione (GSH) and tris cysteine zinc finger proteins in a PbS3 coordination environment

²⁰⁷Pb NMR spectroscopy can be used to monitor the binding of Pb(II) to thiol rich biological small molecules such as glutathione and to zinc finger proteins. The UV/visible (UV/Vis) absorption band centered at 334 nM and the observed ²⁰⁷Pb signal in ²⁰⁷Pb NMR (δ ~ 5750 ppm) indicate that glutathione binds Pb(II) in a trigonal pyramidal geometry (PbS₃) at pH 7.5 or higher with a 1:3 molar ratio of Pb(II) to GSH. While previous studies using UV/Vis and extended X-ray absorption fine structure (EXAFS) spectroscopy were interpreted to show that the zinc binding domain from HIV nucleocapsid protein (HIV-CCHC) binds Pb(II) in a single PbS₃ environment, the more sensitive ²⁰⁷Pb NMR spectra (at pH 7.0, 1:1 molar ratio) provide compelling evidence for the presence of two PbS₃ structures (δ - 5790 and 5744 ppm), one of which is more stable at high temperatures. It has previously been proposed that the HIV-CCHH peptide does not fold properly to afford a PbS₂N motif, because histidine does not bind to Pb(II). These predictions are confirmed by the present studies. These results demonstrate the applicability of ²⁰⁷Pb NMR to biomolecular structure determination in proteins with cysteine binding sites for the first time.     

Genome editing with CompoZr custom zinc finger nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes. Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA. Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency. While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site. The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator's cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.     

Structure based design of protein linkers for zinc finger nuclease

Zinc finger nucleases are a promising tool to edit DNA in many biological applications, in particular for gene knockout. Despite many efforts the number of genes that can be effectively targeted with ZFNs remains severely limited, as available constructs cannot address arbitrary gene sequences. Here, we develop a novel concept to significantly enhance the number of DNA sequences that can be targeted by ZFN. Using an efficient computational model, we provide an extensive library of possible linker molecules between individual zinc finger motifs in the construct that can skip up to 10 base pairs between adjacent zinc finger recognition sites in the DNA sequence, which increases the number of genes that can be efficiently targeted by more than an order of magnitude.     

Regulation of zinc transporters by dietary zinc supplement in breast cancer

Zinc is essential for cell growth and is a co-factor for more than 300 enzymes, representing over 50 different enzyme classes. Two gene families have been identified involved in zinc homeostasis. ZnT transporters reduce intracellular zinc while ZIP transporters increase intracellular zinc. Previous studies have shown that zinc concentration in breast cancer tissues is higher than that in normal breast tissues. However, the mechanisms involved and the relations to zinc transporters are still unknown. A series of zinc transporters are characterized in this article and several of that are emphasized in view of their unique tissue-specific expressions. Established human breast cancer in a nude mice model is used. With a dietary zinc supplement treatment, ZnT-1 mRNA expression in established human breast cancer is raised by 24%, and is nearly 2 times of that in basal diet. ZIP1, ZIP2 and LIV-1 mRNA are the same between two treatment groups. Moreover, no significant changes of these zinc transporters expressions are found between differential breast cancer cell lines in the nude mice model. This is the first report, which detects the zinc transporters expressions in established human breast cancer in nude mice model. These results lead to the constitutive expression and response to zinc in different tissues. In addition to that, ZnT-1 seems to have played an important role in zinc homeostasis, even in breast cancer.     

Lineage-specific expansion of the zinc finger associated domain ZAD

The zinc finger associated domain (ZAD), present in almost 100 distinct proteins, characterizes the largest subgroup of C2H2 zinc finger proteins in Drosophila melanogaster and was initially found to be encoded by arthropod genomes only. Here, we report that the ZAD was also present in the last common ancestor of arthropods and vertebrates, and that vertebrate genomes contain a single conserved gene that codes for a ZAD-like peptide. Comparison of the ZAD proteomes of several arthropod species revealed an extensive and species-specific expansion of ZAD-coding genes in higher holometabolous insects, and shows that only few ZAD-coding genes with essential functions in Drosophila melanogaster are conserved. Furthermore, at least 50% of the ZAD-coding genes of Drosophila melanogaster are expressed in the female germline, suggesting a function in oocyte development and/or a requirement during early embryogenesis. Since the majority of the essential ZAD coding genes of Drosophila melanogaster were not conserved during arthropod or at least during insect evolution, we propose that the LSE of ZAD-coding genes shown here may provide the raw material for the evolution of new functions that allow organisms to pursue novel evolutionary paths.     

Arsenite interacts selectively with zinc finger proteins containing C3H1 or C4 motifs

Arsenic inhibits DNA repair and enhances the genotoxicity of DNA-damaging agents such as benzo[a]pyrene and ultraviolet radiation. Arsenic interaction with DNA repair proteins containing functional zinc finger motifs is one proposed mechanism to account for these observations. Here, we report that arsenite binds to both CCHC DNA-binding zinc fingers of the DNA repair protein PARP-1 (poly(ADP-ribose) polymerase-1). Furthermore, trivalent arsenite coordinated with all three cysteine residues as demonstrated by MS/MS. MALDI-TOF-MS analysis of peptides harboring site-directed substitutions of cysteine with histidine residues within the PARP-1 zinc finger revealed that arsenite bound to peptides containing three or four cysteine residues, but not to peptides with two cysteines, demonstrating arsenite binding selectivity. This finding was not unique to PARP-1; arsenite did not bind to a peptide representing the CCHH zinc finger of the DNA repair protein aprataxin, but did bind to an aprataxin peptide mutated to a CCHC zinc finger. To investigate the impact of arsenite on PARP-1 zinc finger function, we measured the zinc content and DNA-binding capacity of PARP-1 immunoprecipitated from arsenite-exposed cells. PARP-1 zinc content and DNA binding were decreased by 76 and 80%, respectively, compared with protein isolated from untreated cells. We observed comparable decreases in zinc content for XPA (xeroderma pigmentosum group A) protein (CCCC zinc finger), but not SP-1 (specificity protein-1) or aprataxin (CCHH zinc finger). These findings demonstrate that PARP-1 is a direct molecular target of arsenite and that arsenite interacts selectively with zinc finger motifs containing three or more cysteine residues.     

Selectivity of arsenite interaction with zinc finger proteins

Arsenic is a carcinogenic element also used for the treatment of acute promyelocytic leukemia. The reactivity of proteins to arsenic must be associated with the various biological functions of As. Here, we investigated the selectivity of arsenite to zinc finger proteins (ZFPs) with different zinc binding motifs (C2H2, C3H, and C4). Single ZFP domain proteins were used for the direct comparison of the reactivity of different ZFPs. The binding constants and the reaction rates have been studied quantitatively. Results show that both the binding affinity and reaction rates of single-domain ZFPs follow the trend of C4 > C3H ? C2H2. Compared with the C2H2 motif ZFPs, the binding affinities of C3H and C4 motif ZFPs are nearly two orders of magnitude higher and the reaction rates are approximately two-fold higher. The formation of multi-domain ZFPs significantly enhances the reactivity of C2H2 type ZFPs, but has negligible effects on C3H and C4 ZFPs. Consequently, the reactivities of the three types of multi-domain ZFPs are rather similar. The 2D NMR spectra indicate that the As(III)-bound ZFPs are also unfolded, suggesting that arsenic binding interferes with the function of ZFPs.     

Prediction of DNA-binding specificity in zinc finger proteins

Zinc finger proteins interact via their individual fingers to three base pair subsites on the target DNA. The four key residue positions -1, 2, 3 and 6 on the alpha-helix of the zinc fingers have hydrogen bond interactions with the DNA. Mutating these key residues enables generation of a plethora of combinatorial possibilities that can bind to any DNA stretch of interest. Exploiting the binding specificity and affinity of the interaction between the zinc fingers and the respective DNA can help to generate engineered zinc fingers for therapeutic purposes involving genome targeting. Exploring the structure-function relationships of the existing zinc finger-DNA complexes can aid in predicting the probable zinc fingers that could bind to any target DNA. Computational tools ease the prediction of such engineered zinc fingers by effectively utilizing information from the available experimental data. A study of literature reveals many approaches for predicting DNA-binding specificity in zinc finger proteins. However, an alternative approach that looks into the physico-chemical properties of these complexes would do away with the difficulties of designing unbiased zinc fingers with the desired affinity and specificity. We present a physico-chemical approach that exploits the relative strengths of hydrogen bonding between the target DNA and all combinatorially possible zinc fingers to select the most optimum zinc finger protein candidate.     

Quantitative electrospray ionization mass spectrometry of zinc finger oxidation: the reaction of XPA zinc finger with H(2)O(2)

Oxidation plays an important role in the functioning of zinc fingers (ZFs). Electrospray ionization mass spectrometry (ESI-MS) is a very useful technique to study products of ZF oxidation, but its application has been limited largely to qualitative analysis of reaction products. On the other hand, ESI-MS has been applied successfully on several occasions to determine binding constants in metalloproteins. We used a synthetic 37-residue peptide acetyl-DYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHK-amide (XPAzf), which corresponds to the Cys4 ZF sequence of human nucleotide excision repair protein XPA, to find out whether ESI-MS might be used quantitatively to study ZF reaction kinetics. For this purpose, we studied oxidation of the Zn(II) complex of XPAzf (ZnXPAzf) by H(2)O(2) using three techniques in parallel: high-performance liquid chromatography (HPLC) of covalent reaction products, 4-(2-pyridylazo)-resorcinol monosodium salt (PAR)-based spectrophotometric zinc release assay, and ESI-MS. Single and double intrapeptide disulfides were detected by ESI-MS to be the sole reaction products. All three techniques yielded independently the same reaction rate, thereby demonstrating that ESI-MS may indeed be used in quantitative kinetic studies of ZF reactions. The comparison of experimental information demonstrated that the formation of the Cys5-Cys8 single disulfide was responsible for zinc release.     

Electrophoretic mobility shift assay of zinc finger proteins: competition for Zn(2+) bound to Sp1 in protocols including EDTA

The electrophoretic mobility shift assay (EMSA) offers a principal method to detect specific DNA-protein interactions. As commonly conducted, the reaction and electrophoresis running buffers contain large concentrations of EDTA. EDTA has large affinity for Zn²⁺ and readily competes with zinc finger peptides for Zn²⁺ resulting in protein unfolding. Nevertheless, EMSA is routinely used to detect zinc finger protein-DNA adducts. This paper examines the chemistry that permits the detection of zinc finger-DNA complexes in the presence of EDTA, using Zn₃-Sp1 and a cognate DNA binding site, GC1. Twice as much adduct was detected when the reaction was conducted in the absence than in the presence of EDTA. The observation of Zn-Sp1-GC1 was shown to depend on three properties: the inertness of Zn-Sp1-GC1 to reaction with EDTA and the comparatively similar rates of reaction of EDTA and GC1 with Zn₃-Sp1 under the conditions of the assay that permit some Zn₃-Sp1-GC1 to form. Inquiring about the mechanism of stabilization of Zn₃-Sp1 by GC1, EDTA readily reacted with Zn₃-Sp1 bound to a non-specific DNA, (polydI-dC). Two structurally similar but oppositely charged chelators, nitrilotriacetate (NTA) and tris-(2-ethylaminoethyl) amine (TREN), that react with free Zn₃-Sp1 failed to compete for zinc bound in the Zn₃-Sp1-GC-1 adduct. On the basis of these, other results indicated that the stability of Zn₃-Sp1-GC-1 has a thermodynamic, not a kinetic origin. It is concluded that the observation of zinc finger proteins in the EMSA rests on a fortuitous set of chemical properties that may vary depending on the structures involved.     

Recovery of adsorbed zinc in acid soils as influenced by prior phosphorus applications

Summary Five acid soils of Hawaii, having histories of heavy P applications were equilibrated with graded quantities of Zn. Amounts of adsorbed Zn were extracted with a single extraction of 0.005M DTPA. The data indicated that most of the added Zn was in available form. Prior P applications either had no effect on recovery or slightly increased it. The results substantiated the earlier findings that P-induced Zn deficiency could not be due to precipitation of Zn as insoluble Zn–P compounds in the soils.