序列特异的三锌指多肽的构建及其在大肠杆菌中的表达

在获得单一锌指突变体的基础上,以小鼠转录因子Zif268的三锌指DNA结合区为模板,利用重叠(Over-lap)PCR技术,获得了关键氨基酸位点同时突变的三锌指突变体ZF123、2ZF123。ZF123、2ZF123分别克隆进pUC-18质粒,序列测定正确后,以pGEX-2T为表达质粒,在大肠杆菌JM109中实现了功能性的表达。经SDS-PAGE分析,表达出了分子量34.0kD的融合蛋白,扫描分析其含量在20%左右。菌体经超声波破碎后,对可溶性融合蛋白进行了纯化得到了游离的目的蛋白,为进一步的DNA结合特性分析、杂交转录因子的构建等奠定了基础。     

Multiconnection of identical zinc finger: implication for DNA binding affinity and unit modulation of the three zinc finger domain

Cys(2)-His(2)-type zinc finger proteins have a tandemly repeated array structure consisting of independent finger modules. They are expected to elevate the DNA binding affinity and specificity by increasing the number of finger modules. To investigate the relation between the number and the DNA binding affinity of the zinc finger, we have designed the two- to four-finger peptides by connecting the central zinc finger (finger 2) of Sp1 with the canonical linker sequence, Thr-Gly-Glu-Lys-Pro. Gel mobility shift assays reveal that the cognate three- and four-finger peptides, Sp1(zf222) and Sp1(zf2222), strongly bind to the predicted target sequences, but the two-finger peptide, Sp1(zf22), does not. Of special interest is the fact that the dissociation constant for Sp1(zf2222) binding to the target DNA is comparable to that for Sp1(zf222). The methylation interference, DNase I and hydroxyl radical footprintings, and circular permutation analyses demonstrate that Sp1(zf2222) binds to its target site with three successive zinc fingers and the binding of the fourth zinc finger is inhibited by DNA bending induced by the binding of the three-finger domain. The present results strongly indicate that the zinc finger protein binds to DNA by the three-finger domain as one binding unit. In addition, this information provides the basis for the design of a novel multifinger protein with high affinity and specificity for long DNA sequences, such as chromosomal DNAs.     

Total synthesis of artificial zinc-finger proteins: Problems and perspectives

The total synthesis of a peptide segment corresponding to the DNA-binding segment of Sp1 (positions 532-623) using a native chemical ligation approach is described. The folding of the synthetic segment in the presence of Zn(II) gave a zinc-coordinated protein. The dissociation constant (K(D)) for the DNA binding of the resulting protein, determined by a gel mobility shift assay, was 130 nM, almost nine times higher than that of the genetically prepared protein. However, methylation interference assay showed an identical sequence specificity of both proteins in DNA recognition. The chemical ligation method to connect the respective zinc-finger units was also accomplished. Successive ligation between a cysteine-containing peptide segment and a chloroacetylated peptide segment gave an artificial three-finger protein, which corresponds to the above DNA-binding segment of Sp1. However, this protein failed to bind DNA, even at 1.25 mM. Assessment of their folding structure based on the absorption spectra of their Co(II) complexes showed that the linker design to connect the respective finger units is critical for the proper folding of the proteins as well as the occurrence of the DNA-binding function.     

Enhanced cleavage of double-stranded DNA by artificial zinc-finger nuclease sandwiched between two zinc-finger proteins

To enhance DNA cleavage by zinc-finger nucleases (ZFNs), we sandwiched a DNA cleavage enzyme with two artificial zinc-finger proteins (AZPs). Because the DNA between the two AZP-binding sites is cleaved, the AZP-sandwiched nuclease is expected to bind preferentially to a DNA substrate rather than to cleavage products and thereby cleave it with multiple turnovers. To demonstrate the concept, we sandwiched a staphylococcal nuclease (SNase), which cleaves DNA as a monomer, between two three-finger AZPs. The AZP-sandwiched SNase cleaved large amounts of dsDNA site-specifically. Such multiple-turnover cleavage was not observed with nucleases that possess a single AZP. Thus, AZP-sandwiched nucleases will further refine ZFN technology.     

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.     

High-resolution solution structure of two members of a conformationally homogeneous combinatorial peptide library based on the classical zinc-finger motif

Summary We describe the high-resolution structure by NMR of two peptides that belong to a combinatorial library based on the zinc-finger motif. The library represents, to the best of our knowledge, the first example of a conformationally homogeneous peptide library and was obtained by introducing random residues in five positions of the -helical portion of a 26-residue consensus peptide (CP1) belonging to the Cys²-Hys² zinc-finger family. The result was shown to be a highly homogeneous -helical library (Bianchi et al., 1995). The structures of the parent compound (CP1) and of a representative member (CP1m) that was selected by screening the library with a monoclonal antibody are compared in detail as an example of the very high stability of the zinc-finger scaffold upon sequence variability. The two peptides exhibit an extremely high degree of structural similarity. The use of this type of conformationally constrained combinatorial library might represent a step forward in the design of peptidomimetics, as it considerably accelerates the process of the identification of the spatial relationship among the pharmacophoric groups.Abbreviations t-Bu tert-butyloxycarbonyl - Fmoc 9-fluorenylmethoxycarbonyl     

Phosphopeptides designed for 5-methylcytosine recognition

An artificial phosphopeptide has been developed through rational design of the interaction with 5-methylcytosine in duplex DNA. The peptide consists of two tandem zinc finger motifs, in one of which the glutamate was replaced with a phosphotyrosine, the phosphotyrosine in the peptide being effective for methylcytosine selectivity of DNA binding. The flexible modulation of the target methylated sequence by rearrangement of zinc finger peptides is possible, and the phosphopeptide provided us an important hint for expansion of the codes for the interactions of zinc fingers with DNA to methylated DNA sequences. The fluorescence-labeled phosphopeptide provided information on the methylation status of genomic DNA through fluorescence anisotropy after a 10 min incubation.     

Recognition of unmodified histone H3 by the first PHD finger of bromodomain-PHD finger protein 2 provides insights into the regulation of histone acetyltransferases monocytic leukemic zinc-finger protein (MOZ) and MOZ-related factor (MORF)

MOZ (monocytic leukemic zinc-finger protein) and MORF (MOZ-related factor) are histone acetyltransferases important for HOX gene expression as well as embryo and postnatal development. They form complexes with other regulatory subunits through the scaffold proteins BRPF1/2/3 (bromodomain-PHD (plant homeodomain) finger proteins 1, 2, or 3). BRPF proteins have multiple domains, including two PHD fingers, for potential interactions with histones. Here we show that the first PHD finger of BRPF2 specifically recognizes the N-terminal tail of unmodified histone H3 (unH3) and report the solution structures of this PHD finger both free and in complex with the unH3 peptide. Structural analysis revealed that the unH3 peptide forms a third antiparallel β-strand that pairs with the PHD1 two-stranded antiparallel β-sheet. The binding specificity was determined primarily through the recognition of arginine 2 and lysine 4 of the unH3 by conserved aspartic acids of PHD1 and of threonine 6 of the unH3 by a conserved asparagine. Isothermal titration calorimetry and NMR assays showed that post-translational modifications such as H3R2me2as, H3T3ph, H3K4me, H3K4ac, and H3T6ph antagonized the interaction between histone H3 and PHD1. Furthermore, histone binding by PHD1 was important for BRPF2 to localize to the HOXA9 locus in vivo. PHD1 is highly conserved in yeast NuA3 and other histone acetyltransferase complexes, so the results reported here also shed light on the function and regulation of these complexes.     

Contacts between 5 S DNA and Xenopus TFIIIA identified using 5-azido-2'-deoxyuridine-substituted DNA

A highly photosensitive analogue of thymidine, 5-azidodeoxyuridine 5'-triphosphate, has been incorporated into 61-base pair (bp) DNA fragments corresponding to the central region of Xenopus somatic-type 5 S RNA genes such that 5-azidodeoxyuridine replaces some or all T residues in either the coding or noncoding strand of the TFIIIA binding site. Photolysis of TFIIIA.DNA complexes formed with these probes results in efficient, sequence-specific cross-linking to the Zn-finger protein providing direct evidence that this class of proteins have contacts in the major groove of their target sequence. Of the 20 T residues present in the 61-bp probes, greater than 90% of the cross-linking occurs from two sites in the 5 S RNA gene corresponding to T residues at positions 84 and 88 in the noncoding and coding strands, respectively. Digestion by V8 protease of the complex formed with the noncoding strand probe releases peptides not bound to the DNA. Amino acid sequence analysis of the remaining, cross-linked peptides indicates the region including zinc-finger 2 plus the finger 2-3 linker is in contact with position 84. The linker region between fingers 5 and 6 is also in close proximity to the major groove somewhere upstream from position 84.     

The DNA-binding domain of the gene regulatory protein AreA extends beyond the minimal zinc-finger region conserved between GATA proteins

The AreA protein of Aspergillus nidulans regulates the activity of over 100 genes involved in the utilisation of nitrogen, and has a limited region of homology with the vertebrate family of GATA proteins around a zinc finger (Zf) motif. A 66 amino acid (a.a.) residue fragment (Zf(66)) corresponding to the zinc finger, a 91 a.a fragment (Zf(91)) containing an additional 25 a.a. at the C-terminus, and a much larger 728 a.a. sequence (3'EX) corresponding to the 3'exon have been over-expressed as fusion proteins in E. coli and purified. The DNA-protein complexes formed by these proteins have been examined by gel retardation analysis. The 91 a.a. protein forms a discrete shifted species with a GATA-containing DNA fragment with high affinity (K(d)=0.15 nM), whereas the 66 a.a. protein has very low ( approximately microM) affinity for the same sequence. The results show that the region of AreA required for high affinity DNA binding extends beyond the zinc finger motif that is homologous to GATA-1, requiring in addition a region within the 25 a.a. sequence C-terminal to the zinc finger. Using hydroxyl radical and ethylation interference footprinting, the minimal Zinc finger protein (Zf(66)) shows no appreciable interference effects whereas Zf(91) shows much stronger interference effects, identical to those of the larger protein. These effects extend over sequences up to two nucleotides either side of the GATA site, and indicate contacts additional to those observed in the three-dimensional structure of the complex of the minimal zinc-finger protein with DNA. We suggest that these additional contacts are responsible for the enhanced DNA binding affinity of the extended zinc-finger protein Zf(91).