Exploring the recognition of quadruplex DNA by an engineered Cys2-His2 zinc finger protein

We have recently described an engineered zinc finger protein (Gq1) that binds with high specificity to the intramolecular G-quadruplex formed by the human telomeric sequence 5'-(GGTTAG)(5)-3', and that inhibits the activity of the enzyme telomerase in vitro. Here we report site-directed mutagenesis, biophysical, and molecular modeling studies that provide new insights into quadruplex recognition by the zinc finger scaffold. We show that any one finger of Gq1 can be replaced with the corresponding finger of Zif268, without significant loss of quadruplex affinity or quadruplex versus duplex discrimination. Replacement of two fingers, with one being finger 2, of Gq1 by Zif268 results in significant impairment of quadruplex recognition and loss of discrimination. Molecular modeling suggests that the zinc fingers of Gq1 can bind to the human parallel-stranded quadruplex structure in a stable arrangement, whereas Zif268-quadruplex models show significantly weaker binding energy. Modeling also suggests that an important role of the key protein finger residues in the Gq1-quadruplex complex is to maintain Gq1 in an optimum conformation for quadruplex recognition.     

The RING finger motif of photomorphogenic repressor COP1 specifically interacts with the RING-H2 motif of a novel Arabidopsis protein.

The constitutive photomorphogenic 1 (COP1) protein of Arabidopsis functions as a molecular switch for the seedling developmental fates: photomorphogenesis under light conditions and skotomorphogenesis in darkness. The COP1 protein contains a cysteine-rich zinc-binding RING finger motif found in diverse groups of regulatory proteins. To understand the role of the COP1 RING finger in mediating protein-protein interaction, we have performed a yeast two-hybrid screen and isolated a novel protein with a RING-H2 motif, a variant type of the RING finger. This protein, designated COP1 Interacting Protein 8 (CIP8), is encoded by a single copy gene and localized to cytosol in a transient assay. In addition to the RING-H2 motif, the predicted protein has a C4 zinc finger, an acidic region, a glycine-rich cluster, and a serine-rich cluster. The COP1 RING finger and the CIP8 RING-H2 domains are sufficient for their interaction with each other both in vitro and in yeast, whereas neither motif displayed significant self-association. Moreover, site-directed mutagenesis studies demonstrated that the expected zinc-binding ligands of the RING finger and RING-H2 fingers are essential for their interaction. Our findings indicate that the RING finger motif, in this case, serves as autonomous protein-protein interaction domain. The allele specific effect of cop1 mutations on the CIP8 protein accumulation in seedlings indicates that its stability in vivo is dependent on the COP1 protein.     

The zinc finger motif of Escherichia coli RecQ is implicated in both DNA binding and protein folding

The RecQ family of DNA helicases has been shown to be important for the maintenance of genomic integrity. Mutations in human RecQ genes lead to genomic instability and cancer. Several RecQ family of helicases contain a putative zinc finger motif of the C4 type at the C terminus that has been identified in the crystalline structure of RecQ helicase from Escherichia coli. To better understand the role of this motif in helicase from E. coli, we constructed a series of single mutations altering the conserved cysteines as well as other highly conserved residues. All of the resulting mutant proteins exhibited a high level of susceptibility to degradation, making functional analysis impossible. In contrast, a double mutant protein in which both cysteine residues Cys397 and Cys400 in the zinc finger motif were replaced by asparagine residues was purified to homogeneity. Slight local conformational changes were detected, but the rest of the mutant protein has a well defined tertiary structure. Furthermore, the mutant enzyme displayed ATP binding affinity similar to the wild-type enzyme but was severely impaired in DNA binding and in subsequent ATPase and helicase activities. These results revealed that the zinc finger binding motif is involved in maintaining the integrity of the whole protein as well as DNA binding. We also showed that the zinc atom is not essential to enzymatic activity.     

The zinc finger antiviral protein directly binds to specific viral mRNAs through the CCCH zinc finger motifs

The zinc finger antiviral protein (ZAP) is a recently isolated host antiviral factor. It specifically inhibits the replication of Moloney murine leukemia virus (MLV) and Sindbis virus (SIN) by preventing the accumulation of viral RNA in the cytoplasm. For this report, we mapped the viral sequences that are sensitive to ZAP inhibition. The viral sequences were cloned into a luciferase reporter and analyzed for the ability to mediate ZAP-dependent destabilization of the reporter. The sensitive sequence in MLV was mapped to the 3' long terminal repeat; the sensitive sequences in SIN were mapped to multiple fragments. The fragment of SIN that displayed the highest destabilizing activity was further analyzed by deletion mutagenesis for the minimal sequence that retained the activity. This led to the identification of a fragment of 653 nucleotides. Any further deletion of this fragment resulted in significantly lower activity. We provide evidence that ZAP directly binds to the active but not the inactive fragments. The CCCH zinc finger motifs of ZAP play important roles in RNA binding and antiviral activity. Disruption of the second and fourth zinc fingers abolished ZAP's activity, whereas disruption of the first and third fingers just slightly lowered its activity.     

Zinc is required for folding and binding of a single zinc finger to DNA

A synthetic peptide corresponding to zinc finger 31 of the Xenopus protein Xfin adopts a folded conformation in the presence of zinc. The same peptide in the absence of zinc is not folded in a stable tertiary conformation, as determined by NMR. Binding experiments have shown that the peptide binds non-specifically to DNA only in the presence of zinc. Moreover, competitive DNA binding experiments indicate interaction with 3.9 +/- 0.4 base pairs.     

Expression analysis of an Arabidopsis C2H2 zinc finger protein gene

C₂H₂ zinc finger protein genes encode nucleic acid-binding proteins involved in the regulation of gene activity. AtZFP1 (Arabidopsis thaliana zinc finger protein 1) is one member of a small family of C₂H₂ zinc finger-encoding sequences previously characterized from Arabidopsis. The genomic sequence corresponding to the AtZFP1 cDNA has been determined. Molecular analysis demonstrates that AtZFP1 is a unique, intronless gene which encodes a 1100 nucleotides mRNA highly expressed in roots and stems. A construct in which 2.5 kb of AtZFP1 upstream sequences is linked to the -glucuronidase gene was introduced into Arabidopsis by Agrobacterium-mediated transformation of roots. Histochemical analysis of transgenic Arabidopsis carrying the AtZFP1 promotor:-glucuronidase fusion shows good correlation with RNA blot hybridization analysis. This transgenic line will be a useful tool for analyzing the regulation of AtZFP1 to further our understanding of its function.     

The C-terminal zinc finger of UvrA does not bind DNA directly but regulates damage-specific DNA binding

In prokaryotic nucleotide excision repair, UvrA recognizes DNA perturbations and recruits UvrB for the recognition and processing steps in the reaction. One of the most remarkable aspects of UvrA is that it can recognize a wide range of DNA lesions that differ in chemistry and structure. However, how UvrA interacts with DNA is unknown. To examine the role that the UvrA C-terminal zinc finger domain plays in DNA binding, an eleven amino acid deletion was constructed (ZnG UvrA). Biochemical characterization of the ZnG UvrA protein was carried out using UvrABC DNA incision, DNA binding and ATPase assays. Although ZnG UvrA was able to bind dsDNA slightly better than wild-type UvrA, the ZnG UvrA mutant only supported 50-75% of wild type incision. Surprisingly, the ZnG UvrA mutant, while retaining its ability to bind dsDNA, did not support damage-specific binding. Furthermore, this mutant protein only provided 10% of wild-type Bca UvrA complementation for UV survival of an uvrA deletion strain. In addition, ZnG UvrA failed to stimulate the UvrB DNA damage-associated ATPase activity. Electrophoretic mobility shift analysis was used to monitor UvrB loading onto damaged DNA with wild-type UvrA or ZnG UvrA. The ZnG UvrA protein showed a 30-60% reduction in UvrB loading as compared with the amount of UvrB loaded by wild-type UvrA. These data demonstrate that the C-terminal zinc finger of UvrA is required for regulation of damage-specific DNA binding.     

The Dbf4 motif C zinc finger promotes DNA replication and mediates resistance to genotoxic stress

The Dbf4/Cdc7 kinase (DDK) plays an essential role in stimulating DNA replication by phosphorylating subunits of the Mcm2-7 helicase complex at origins. This kinase complex is itself phosphorylated and removed from chromatin in a Rad53-dependent manner when an S phase checkpoint is triggered. Comparison of Dbf4 sequence across a variety of eukaryotic species has revealed three conserved regions that have been termed motifs N, M and C. The most highly conserved of the three, motif C, encodes a zinc finger, which are known to mediate protein-protein and protein-DNA interactions. Mutation of conserved motif C cysteines and histidines disrupted the association of Dbf4 with ARS1 origin DNA and Mcm2, but not other known ligands including Cdc7, Rad53 or the origin recognition complex subunit Orc2. Furthermore, these mutations impaired the ability of Dbf4 to phosphorylate Mcm2. Budding yeast strains for which the single genomic DBF4 copy was replaced with these motif C mutant alleles were compromised for entry into and progression through S phase, indicating that the observed weakening of the Mcm2 interaction prevents DDK from efficiently stimulating the initiation of DNA replication. Following initiation, Mcm2-7 migrates with the replication fork. Interestingly, the motif C mutants were sensitive to long-term, but not short-term exposure to the genotoxic agents hydroxyurea and methyl methanesulfonate. These results support a model whereby DDK interaction with Mcm2 is important to stabilize and/or restart replication forks during conditions where a prolonged S-phase checkpoint is triggered.     

The RdgC protein employs a novel mechanism involving a finger domain to bind to circular DNA

The DNA-binding protein RdgC has been identified as an inhibitor of RecA-mediated homologous recombination in Escherichia coli. In Neisseria species, RdgC also has a role in virulence-associated antigenic variation. We have previously solved the crystal structure of the E. coli RdgC protein and shown it to form a toroidal dimer. In this study, we have conducted a mutational analysis of residues proposed to mediate interactions at the dimer interfaces. We demonstrate that destabilizing either interface has a serious effect on in vivo function, even though a stable complex with circular DNA was still observed. We conclude that tight binding is required for inhibition of RecA activity. We also investigated the role of the RdgC finger domain, and demonstrate that it plays a crucial role in the binding of circular DNA. Together, these data allow us to propose a model for how RdgC loads onto DNA. We discuss how RdgC might inhibit RecA-mediated strand exchange, and how RdgC might be displaced by other DNA metabolism enzymes such as polymerases and helicases.     

The contribution of a zinc finger motif to the function of yeast ribosomal protein YL37a

Eukaryotic ribosomes have a large number of proteins but the exact nature of their contribution to the structure and to the function of the particle is not known. Of the 78 proteins in yeast ribosomes, six have zinc finger motifs of the C2-C2 variety. Both genes encoding the essential yeast ribosomal protein YL37a, which has such a zinc finger motif, were disrupteXXPd. The double deletion, which is lethal, can be rescued with a plasmid-encoded copy of a YL37a gene. Mutations were constructed in a plasmid-encoded copy of YL37a; the mutations caused the cysteine residues in the motif (at positions 39, 42, 57 and 60) to be replaced, one at a time, with serine. The cysteine residue at position 39, the first of the four in the motif, is essential for the function of YL37a, since a C39S mutation did not complement the null phenotype. However, plasmids encoding variants with C42S, C57S, or C60S mutations in the zinc finger motif were able to rescue the null mutant. YL37a binds zinc, but none of the mutant proteins, C39S, C42S, C57S, or C60S, was able to bind the metal. Thus, all four cysteine residues are essential for the binding of zinc; only one, C39, is essential for the function of the ribosomal protein.     

Structure of a designed dimeric zinc finger protein bound to DNA

Proteins that employ dimerization domains to bind cooperatively to DNA have a number of potential advantages over monomers with regards to gene regulation. Using a combination of structure-based design and phage display, a dimeric Cys(2)His(2) zinc finger protein has been created that binds cooperatively to DNA via an attached leucine zipper dimerization domain. This chimera, derived from components of Zif268 and GCN4, displayed excellent DNA-binding specificity, and we now report the 1.5 A resolution cocrystal structure of the Zif268-GCN4 homodimer bound to DNA. This structure shows how phage display has annealed the DNA binding and dimerization domains into a single functional unit. Moreover, this chimera provides a potential platform for the creation heterodimeric zinc finger proteins that can regulate a desired target gene through cooperative DNA recognition.