一种特异识别SV40启动子的人工转录因子的构建

转录因子是真核表达调控中非常重要的一类反式作用因子,通常由DNA结合域与效应域两部分组成,而锌指结构是DNA结合域的常见组成单元。人工转录因子就是基于转录因子的这种结构特点,人为地选择针对特定序列的DNA结合域与具有特定作用的效应域组合而成。利用噬菌体展示技术,筛选到与SV40启动子上9bp序列特异结合的锌指结构,再连接KOX1的KRAB域构建了一种人工转录因子。转染实验表明它对SV40下游的报告基因的表达有很显著的抑制作用。     

人工转录因子研究进展

转录因子是真核表达调控中非常重要的一类反式作用因子,通常由DNA结合结构域与效应结构域两部分组成,研究发现这两个结构域可以各自独立发生作用。基于转录因子的这种结构特点,可以人为地选择针对特定序列的DNA结合结构域与具有特定作用的效应结构域构建人工转录因子。目前人工转录因子的DNA结合结构域多为C₂H₂ 型锌指结构,每一个锌指单元由大约30个氨基酸组成,识别DNA双螺旋大沟中相连的3bp序列,并可通过氢键作用与相应的碱基结合;多个锌指可以串联成簇,从而识别并结合较长的DNA序列区域。常见的人工转录因子的效应结构域有激活结构域以及抑制结构域,不同的效应结构域赋予人工转录因子不同的功能。目前人工转录因子已经在基础研究、药物设计以及基因治疗等领域得到了广泛的应用。     

DNA-Binding ability of GAGA zinc finger depends on the nature of amino acids present in the beta-hairpin

The GAGA factor of Drosophila melanogaster uses a single Cys2-His2-type zinc finger for specific DNA binding. Comparative sequence alignment of the GAGA zinc finger core with other structurally characterized zinc fingers reveals that the beta-hairpin of the GAGA zinc finger prefers amino acids with an aliphatic side-chain different from those of other zinc fingers. To probe the substitution effect of aromatic amino acids in the beta-hairpin on the DNA binding, three mutant peptides were designed by substituting consensus phenylalanine, an aromatic amino acid, at key positions in the beta-hairpin region. The metal-binding and the overall fold of the mutant peptides are very similar to those of the wild-type as shown by UV-vis absorption spectroscopy and circular dichroism spectroscopy. However, the gel mobility shift assay and isothermal calorimetric studies demonstrated that none of the mutants are able to bind the cognate DNA substrate, although the mutation is confined only to the beta-hairpin region. The present results suggest that the nature of the amino acids in the beta-hairpin plays an important role in the DNA-binding of the GAGA factor protein.     

Site-specific DNA cleavage by artificial zinc finger-type nuclease with cerium-binding peptide

The addition of a new function to native proteins is one of the most attractive protein-based designs. In this study, we have converted a C(2)H(2)-type zinc finger as a DNA-binding motif into a novel zinc finger-type nuclease by connecting two distinct zinc finger proteins (Sp1 and GLI) with a functional linker possessing DNA cleavage activity. As a DNA cleavage domain, we chose an analogue of the metal-binding loop (12 amino acid residues), peptide P1, which has been reported to exhibit a strong binding affinity for a lanthanide ion and DNA cleavage ability in the presence of Ce(IV). Our newly designed nucleases, Sp1(P1)GLI and Sp1(P1G)GLI, can strongly bind to a lanthanide ion and show a unique DNA cleavage pattern, in which certain positions between the two DNA-binding sites are specifically cleaved. The present result provides useful information for expanding the design strategy for artificial nucleases.     

Two DNA-binding and nick recognition modules in human DNA ligase III

Human DNA ligase III contains an N-terminal zinc finger domain that binds to nicks and gaps in DNA. This small domain has been described as a DNA nick sensor, but it is not required for DNA nick joining activity in vitro. In light of new structural information for mammalian ligases, we measured the DNA binding affinity and specificity of each domain of DNA ligase III. These studies identified two separate, independent DNA-binding modules in DNA ligase III that each bind specifically to nicked DNA over intact duplex DNA. One of these modules comprises the zinc finger domain and DNA-binding domain, which function together as a single DNA binding unit. The catalytic core of ligase III is the second DNA nick-binding module. Both binding modules are required for ligation of blunt ended DNA substrates. Although the zinc finger increases the catalytic efficiency of nick ligation, it appears to occupy the same binding site as the DNA ligase III catalytic core. We present a jackknife model for ligase III that posits conformational changes during nick sensing and ligation to extend the versatility of the enzyme.     

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.     

Human herpesvirus 6B origin-binding protein: DNA-binding domain and consensus binding sequence.

We previously demonstrated by a DNA-binding assay that the human herpesvirus 6B (HHV-6B) replication origin has a structure similar to those of alphaherpesviruses, although the HHV-6B and herpes simplex virus type 1 (HSV-1) origin-binding proteins (OBPs) and origins are not interchangeable. Here we describe additional properties of the interaction between HHV-6B OBP and the HHV-6B origin. Competitive electrophoretic mobility shift assays (EMSAs) with DNA duplexes containing single-base alterations allowed deduction of a consensus DNA sequence for HHV-6B-specific OBP binding, YGWYCWCCY, where Y is T or C and W is T or A, while that for HSV-1-specific binding was reported to be YGYTCGCACT. By EMSA, the HHV-6B OBP DNA-binding domain was mapped to a segment containing amino acids 482 to 770. However, in Southwestern (protein-DNA) blotting, the region sufficient for the DNA binding encompassed only amino acids 657 to 770. Similarly, Southwestern blotting showed that amino acids 689 to 851 of HSV-1 OBP had HSV-1 origin-binding activity, although this region was insufficient for origin binding in the EMSA. Although the longer DNA-binding domains identified by EMSA have marginal overall homology among HHV-6B and alphaherpesvirus OBP homologs, the smaller regions sufficient for the binding observed by Southwestern blotting have significant similarity. From these results, we propose a hypothesis that the DNA-binding domain of herpesvirus OBPs consists of two subdomains, one containing a conserved motif that contacts DNA directly, and another, less well conserved, that may modulate either the conformation or accessibility of the binding domain.