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PubMed1995

Transplantation of a 17-amino acid alpha-helical DNA-binding domain into an antibody molecule confers sequence-dependent DNA recognition.

Recombinant antibodies capable of sequence-specific interactions with nucleic acids represent a class of DNA- and RNA-binding proteins with potential for broad application in basic research and medicine. We describe the rational design of a DNA-binding antibody, Fab-Ebox, by replacing a variable segment of the immunoglobulin heavy chain with a 17-amino acid domain derived from TFEB, a class B basic helix-loop-helix protein. DNA-binding activity was studied by electrophoretic mobility-shift assays in which Fab-Ebox was shown to form a specific complex with DNA containing the TFEB recognition motif (CACGTG). Similarities were found in the abilities of TFEB and Fab-Ebox to discriminate between oligodeoxyribonucleotides containing altered recognition sequences. Comparable interference of binding by methylation of cytosine residues indicated that Fab-Ebox and TFEB both contact DNA through interactions along the major groove of double-stranded DNA. The results of this study indicate that DNA-binding antibodies of high specificity can be developed by using the modular nature of both immunoglobulins and transcription factors.

Amino Acid Sequence↗
PubMed1998

Mutational analysis of the STAT6 SH2 domain.

The SH2 domain of the STAT family of transcription factors is essential for STAT binding to phosphorylated cytoplasmic domains of activated cytokine receptors. Furthermore, the same domain mediates dimerization of activated STAT monomers, a prerequisite for DNA binding by this family of proteins. To identify amino acid residues within the STAT protein that mediate these various interactions, we have carried out an extensive mutational analysis of the Stat6 SH2 domain. Recombinant proteins carrying C-terminal deletions or double alanine substitutions were expressed in mammalian and insect cells and assayed for DNA binding, transcription activation, tyrosine phosphorylation, and the ability to interact with a tyrosine-phosphorylated peptide derived from the interleukin-4 receptor signaling chain. From these studies, we have identified amino acids that are required for both DNA binding and interleukin-4 receptor interaction, as well as residues that when mutated impair only one of the two functions. Our results suggest that the structural homology between the SH2 domain of Stat6 and that of the distantly related Src protein may be higher than predicted on the basis of primary amino acid sequence comparisons. However, the two types of SH2 domains may differ at their C-terminal ends.

Alanine↗
PubMed1999

Interaction of cationic alpha-helical peptide with phosphorothioate DNA hybrid.

Synthetic amphiphilic alpha-helix peptides were found to bind to stabilize double or triple stranded DNA. The stabilization effect was significant for cationic alpha-helix peptides which indicated the importance of electrostatic interaction of positive charge of peptide and negative charge of DNA. It should be also pointed out that hybrid double or triple helical complexes containing phosphorothioate oligonucleotide were stabilized to a larger extent respect to phosphodiester oligonucleotides. Since it was shown that cationic amphiphilic alpha-helix peptide accelerate membrane permeability of DNA, the present study can provide a solution for the problems of antisense or triplex oligonucleotide in their practical application.

Amino Acid Sequence↗
PubMed2002

Exportin-5 mediates nuclear export of minihelix-containing RNAs.

The adenovirus VA1 RNA (VA1), a 160-nucleotide (nt)-long RNA transcribed by RNA polymerase III, is efficiently exported from the nucleus to the cytoplasm of infected cells, where it antagonizes the interferon-induced antiviral defense system. We recently reported that nuclear export of VA1 is mediated by a cis-acting RNA export motif, called minihelix, that comprises a double-stranded stem (>14 nt) with a base-paired 5' end and a 3-8-nt protruding 3' end. RNA export mediated by the minihelix motif is Ran-dependent, which indicates the involvement of a karyopherin-related factor (exportin) that remained to be determined. Here we show using microinjection in Xenopus laevis oocytes that VA1 is transported to the cytoplasm by exportin-5, a nuclear transport factor for double-stranded RNA binding proteins. Gel retardation assays revealed that exportin-5 directly interacts with VA1 RNA in a RanGTP-dependent manner. More generally, in vivo and in vitro competition experiments using various VA1-derived, but also artificial and cellular, RNAs lead to the conclusion that exportin-5 preferentially recognizes and transports minihelix motif-containing RNAs.

Active Transport, Cell Nucleus↗
PubMed2005

Differential binding of Escherichia coli O157:H7 to alfalfa, human epithelial cells, and plastic is mediated by a variety of surface structures.

Escherichia coli O157:H7 carried on plant surfaces, including alfalfa sprouts, has been implicated in food poisoning and outbreaks of disease in the United States. Adhesion to cell surfaces is a key component for bacterial establishment and colonization on many types of surfaces. Several E. coli O157:H7 surface proteins are thought to be important for adhesion and/or biofilm formation. Therefore, we examined whether mutations in several genes encoding potential adhesins and regulators of adherence have an effect on bacterial binding to plants and also examined the role of these genes during adhesion to Caco-2 cells and during biofilm formation on plastic in vitro. The genes tested included those encoding adhesins (cah, aidA1, and ompA) and mediators of hyperadherence (tdcA, yidE, waaI, and cadA) and those associated with fimbria formation (csgA, csgD, and lpfD2). The introduction of some of these genes (cah, aidA1, and csg loci) into an E. coli K-12 strain markedly increased its ability to bind to alfalfa sprouts and seed coats. The addition of more than one of these genes did not show an additive effect. In contrast, deletion of one or more of these genes in a strain of E. coli O157:H7 did not affect its ability to bind to alfalfa. Only the absence of the ompA gene had a significant effect on binding, and the plant-bacterium interaction was markedly reduced in a tdcA ompA double mutant. In contrast, the E. coli O157:H7 ompA and tdcA ompA mutant strains were only slightly affected in adhesion to Caco-2 cells and during biofilm formation. These findings suggest that some adhesins alone are sufficient to promote binding to alfalfa and that they may exist in E. coli O157:H7 as redundant systems, allowing it to compensate for the loss of one or more of these systems. Binding to the three types of surfaces appeared to be mediated by overlapping but distinct sets of genes. The only gene which appeared to be irreplaceable for binding to plant surfaces was ompA.

Adhesins, Bacterial↗
PubMed1988

Partial purification and characterization of a cellular protein that binds to the SV40 core origin of DNA replication.

A monkey cell factor that interacts specifically with double- and single-stranded DNA sequences in the early domain of the simian virus 40 (SV40) core origin of replication was identified using gel-retention assays. The protein was enriched over 1200-fold using ion-exchange and affinity chromatography on single-strand DNA cellulose. Binding of protein to mutant origin DNA restriction fragments was correlated with replication activity of the mutant DNAs. Exonuclease footprint experiments on single-stranded DNA revealed prominent pause sites in the early domain of the core origin. The results suggest that this cellular protein may be involved in SV40 DNA replication.

Base Sequence↗
PubMed2005

Antitumor polycyclic acridines. Part 16. Triplex DNA as a target for DNA-binding polycyclic acridine derivatives.

Triple-stranded DNA structures have been implicated in a number of major biological processes, including the transcription and translation of a number of genes, as well as in the interaction of DNA with a number of proteins. Furthermore, antigene therapies under development are based on the recognition and binding of a single oligonucleotide strand to a double-stranded sequence, thus forming a triple helix. Triplex DNA formation is a relatively weak and temporary phenomenon; therefore, molecules that selectively bind to and stabilize triple helices may show a variety of novel biological effects. The biophysical and biological characterization of a series of antitumor polycyclic acridines that bind to triplex DNA is reported. These compounds, whose synthesis has been previously reported, have been tested for their interaction with both purine and pyrimidine type triple helices and compared with the relevant double-stranded DNA. As a pyrimidine triplex model we have used the T*AT sequence, which we have compared with the AT duplex, whereas the purine triplex oligonucleotide d[G3A4G3]*d[G3A4G3].d[C3T4C3] has been compared with the duplex d[G3A4G3].d[C3T4C3]. The compounds demonstrate various degrees of preferential binding to triplex DNA over normal duplex DNA, as measured by UV, fluorescence, circular dichroism, and thermal denaturation. Tri-substituted acridine derivatives demonstrated the highest affinity and ability to stabilize triplex DNA structures. Furthermore, structure/affinity analysis gives insights into the structural features that optimize affinity and selectivity for triplex DNA, and may play a role in their profile of antitumor activity.

Acridines↗
PubMed2006

Interactions of quercetin and its lanthane complex with double stranded DNA/RNA and single stranded RNA: spectrophotometric sensing of poly G.

Spectrophotometric titrations revealed that stability of the quercetin/double stranded (ds) DNA or double stranded (ds) RNA non-covalent complexes is significantly higher compared to the quercetin/ss-RNA complexes. This observation can easily be correlated with the significantly larger aromatic surface of base pairs compared to single nucleobases, and it is in good agreement with other experimental data pointing toward intercalative binding mode of quercetin. Fluorescence increase of quercetin induced by ds-RNA is significantly stronger than observed for ds-DNA, offering usage of quercetin as the ds-RNA selective fluorescent probe. Also, addition of poly G yielded more than order of magnitude stronger changes in UV/visible and fluorescence spectrum of quercetin compared to the changes upon addition of poly A and poly U revealing possible usage of quercetin as a powerful spectroscopic probe for poly G sequences. Stability and stoichiometry of lanthane(III)/quercetin complexes in physiologically relevant aqueous media was determined. The interactions of (LaQ)(3+) with double stranded DNA and RNA were significantly different compared to the free quercetin, revealing increase of complex stability and thus significant impact of La(III) in binding of (LaQ)(3+) to polynucleotides. Similar results were observed for interactions of (LaQ)(3+) with single stranded RNA.

Binding Sites↗
PubMed1986

Direct measurement of the interaction between phosphatidylglycerol bilayers in aqueous electrolyte solutions.

Results are presented of force measurements between deposited bilayers of dimyristoylphosphatidyl glycerol (DMPG) at T greater than Tm, and distearoylphosphatidyl glycerol (DSPG) at T less than Tm. Below a bilayer separation of 100 nm, a repulsive double-layer force is measured, which can be explained through the combined screening and binding effect of the counterions in electrolyte solutions of NaCl, HCl, CaCl2, or mixtures of these. The binding of cations to bilayers in the fluid phase (DMPG) appears to be greater than to bilayers in the gel phase (DSPG). At shorter range, below approximately 3 nm, an attractive interaction is measured in solutions containing CaCl2, which was found to be slightly stronger than the theoretically expected van der Waals interaction. No hydration force was observed to exist in solutions containing CaCl2. In NaCl solutions, the measured interbilayer force can completely be accounted for by the electrostatic repulsion, down to a bilayer separation of at least 2 nm, below which no accurate measurements were possible anymore. Parallel measurements on PG monolayers show that the contraction of a DMPG monolayer following addition of CaCl2 is significantly greater than what is predicted from the change in the double-layer free energy alone. This indicates that changes in the lateral interactions between the lipid headgroups probably involve Ca2+-bridge binding and/or a possible dehydration of the lipid headgroups through Ca2+ binding. The results shed new light on both the interbilayer and intrabilayer interactions of PG and identify the possible factors responsible for the morphological behavior of PG aggregates.

Electrolytes↗
PubMed1994

The manganese superoxide dismutase of Escherichia coli K-12 associates with DNA.

Superoxide dismutases (SODs) are vital components in the resistance of aerobic organisms to the toxicity of oxygen. Escherichia coli contains two highly homologous cytoplasmic SODs, a manganese- and an iron-containing enzyme (MnSOD, FeSOD). We previously demonstrated that MnSOD and FeSOD have different physiological functions and that MnSOD is more effective in preventing oxidative damage to DNA. In this report, purified E. coli MnSOD was shown to bind nonspecifically to DNA by electrophoretic mobility shift assay and nitrocellulose-filter binding methodologies. From electrophoretic mobility shift assay, the equilibrium dissociation constants for interaction with a variety of double-stranded and single-stranded oligonucleotides ranged from 1.5 +/- 0.2 to 8.4 +/- 1.3 microM at 20 degrees C. This range of concentrations corresponds to MnSOD concentrations in aerobically grown E. coli. In vivo binding of MnSOD to DNA was supported by colocalization of MnSOD and the E. coli nucleoid in immunoelectron microscopy. Both MnSOD and DNA were inhomogeneously distributed in the cytosol, the concentration of each being higher in the center of the cell and relatively low near the inner membrane. In contrast, there was no evidence for physiologically relevant interaction of FeSOD with DNA. Binding to DNA in vitro was weak, Kd > 40-220 microM, concentrations 7-40 times higher than found in vivo. In addition, the cytoplasmic distribution of FeSOD did not correlate with DNA. FeSOD concentration was higher near the inner membrane and lower in the center of the cytosol. These results demonstrate that E. coli MnSOD associates with DNA in vitro and in vivo. Combined with prior data demonstrating that MnSOD preferentially protects DNA in vivo while an equal enzymatic activity of FeSOD does not (Hopkin, K. A., Papazian, M. A., and Steinman, H. M. (1992) J. Biol. Chem. 267, 24253-24258), our data suggest that E. coli MnSOD acts as a "tethered antioxidant"; association of MnSOD with DNA localizes dismutase activity near a target of oxidative stress and increases protection of DNA from oxidative damage. This model has implications for the therapeutic use of SODs as antioxidants.

Antibodies↗
PubMed2005

Binding of herpes simplex virus-1 US11 to specific RNA sequences.

Herpes simplex virus-1 US11 is a RNA-binding protein with a novel RNA-binding domain. US11 has been reported to exhibit sequence- and conformation-specific RNA-binding, but the sequences and conformations important for binding are not known. US11 has also been described as a double-stranded RNA (dsRNA)-binding protein. To investigate the US11-RNA interaction, we performed in vitro selection of RNA aptamers that bind US11 from a RNA library consisting of >10(14) 80 base sequences which differ in a 30 base randomized region. US11 bound specifically to selected aptamers with an affinity of 70 nM. Analysis of 23 selected sequences revealed a strong consensus sequence. The US11 RNA-binding domain and < or =46 bases of selected RNA containing the consensus sequence were each sufficient for binding. US11 binding protected the consensus motif from hydroxyl radical cleavage. RNase digestions of a selected aptamer revealed regions of both single-stranded RNA and dsRNA. We observed that US11 bound two different dsRNAs in a sequence non-specific manner, but with lower affinity than it bound selected aptamers. The results define a relatively short specific sequence that binds US11 with high affinity and indicate that dsRNA alone does not confer high-affinity binding.

Base Sequence↗
PubMed1996

Relative contribution of polar interactions and conformational compatibility to the binding of neurokinin-1 receptor antagonists.

Based on single residue substitutions, previous studies suggested that Gln165, His197, and His265 of the neurokinin-1 receptor interact directly with many nonpeptide antagonists, including CP-96,345. To further test this model, all three residues have been substituted simultaneously with alanine. The Q165A-H197A-H265A triple mutant bound CP-96,345 and eight analogs with similar affinity (2-20 microM), even though the same series of compounds bound to the wild-type receptor with affinities over a range of 1000-fold. These observations correspond exactly to the prediction of the binding site model. The micromolar binding affinity of all tested CP-96,345 analogs for the triple mutant seems to reflect solely van der Waals interactions, which suggests a significant contribution of conformational compatibility (or shape complementarity) to binding affinity. The primary role of conformational compatibility in ligand binding was consistent with the observation that simply transferring the residues involved in polar interactions with beta2-agonists into the neurokinin-1 receptor did not lead to increased binding affinity for the beta2-agonists. Taken together, these results support a general principle of ligand-receptor binding in which specific polar interactions can take place only if the overall ligand conformation is compatible with the stereochemistry of the binding pocket. In addition, double-residue and triple-residue substitutions, in combination with single-residue substitutions, can provide an alternative route to reveal multiple interactions that may not be detectable by single-residue substitutions and represent a novel approach to examine ligand-receptor interactions in the absence of high-resolution structural data.

Amino Acid Sequence↗
PubMed2000

Role of Mg2+ in the interaction of anticancer antibiotic, chromomycin A3 with DNA: does neutral antibiotic bind DNA in absence of the metal ion?

Antitumor antibiotic, Chromomycin A3 (CHR), inhibits DNA replication and transcription via reversible interaction with double stranded DNA with GC-base specificity. The interaction, at and above physiological pH, requires the presence of bivalent metal ions, such as Mg2+. Anionic antibiotic does not bind DNA in the absence of Mg2+. In this paper we have examined the structural potential of neutral CHR at pH 5.2 to bind DNA in the absence of Mg2+. We have demonstrated the ability of the neutral antibiotic to bind DNA by means of different spectroscopic techniques and evaluated the necessary thermodynamic parameters for elucidation of the molecular basis of recognition. The results are compared with the scenario when Mg2+ is present in the system, because the ultimate aim of these studies is to elucidate the role of Mg2+ in CHR-DNA recognition. Neutral CHR binds to Mg2+ with lesser affinity than its anionic form. Spectroscopic features of the drug and its Mg2+ complex indicate self association of the antibiotic in the absence and presence of Mg2+. GC-base specificity of the drug and its Mg2+ complex are retained at pH 5.2, though the modes of recognition of DNA by the two ligands are different. Minor groove width of DNA plays a role in the accommodation of the ligand(s) during the GC base specific recognition while positive charge of Mg2+ in CHR:Mg2+ complex further facilitates the association. Relatively lower affinity of the neutral drug and its Mg2+ complex for DNA can be ascribed to the self association of these ligands in the absence of DNA.

Animals↗
PubMed1986

Fluorescence of 3-keto-steroids in aqueous solution. Probes for steroid-protein interactions.

The physiologically important 3-keto-steroids are non-fluorescent or only weakly fluorescent in protic as well as in aprotic solvents. In contrast, the 4,6,8(14)-triene-3-one steroids are highly fluorescent in aqueous solution but they do not appreciably fluoresce in other solvents. Evidence is presented that the introduction of double bonds into the skeleton of the 3-keto-steroids leads to a decrease of the energy of the lowest pi-pi* state, bringing this level into the neighbourhood of the non-fluorescent n-pi* state. As a consequence, for two states of approximately the same energy, relatively small perturbations such as those due to solvent interactions, protein binding and micelle formation, will then determine whether a system will fluoresce (pi-pi* state lowest) or not (n-pi* state lowest). When the fluorescent 3-keto-steroids, having three conjugated double bonds, bind to proteins, the fluorescence intensity becomes almost zero, making these compounds useful as probes for steroid-protein interactions. This quenching of the fluorescence is explained by a decrease in energy of the n-pi* state relative to the pi-pi* state of th steroids due to hydrophobic interactions with the proteins.

Biophysical Phenomena↗
PubMed2000

Estrogen receptor protects p53 from deactivation by human double minute-2.

We and others have demonstrated that estrogen receptor alpha (ERalpha) and p53, two important regulatory proteins in breast cancer, bind to each other. In this report, using the glutathione S-transferase pull-down methodology, we show the ligand-independent interaction of ERalpha with the NH2-terminal region of p53, a region known to bind the p300 and human double minute-2 (hdm2) regulatory factors. Furthermore, we have demonstrated that ERalpha is capable of binding hdm2 directly. The interaction of ERalpha and p53 does not interfere with the binding between p53 and hdm2; rather, these proteins form a ternary complex. The effect of ERalpha on the p53-hdm2 regulatory loop has been examined. Our results indicate that ERalpha protects p53 from being deactivated by hdm2. It is evident from these investigations that the ligand-independent protection of p53 by ERalpha is a novel role for this protein in addition to its classic regulatory function as a ligand-inducible transcription factor. This study also describes a new mechanism of cellular regulation of p53 activity.

Breast Neoplasms↗
PubMed2005

Recognition of double-stranded RNA by human toll-like receptor 3 and downstream receptor signaling requires multimerization and an acidic pH.

Studies involving Toll-like receptor 3 (TLR3)-deficient mice suggest that this receptor binds double-stranded RNA. In the present study, we analyzed ligand/receptor interactions and receptor-proximal events leading to TLR3 activation. The mutagenesis approach showed that certain cysteine residues and glycosylation in TLR3 amino-terminal leucine-rich repeats were necessary for ligand-induced signaling. Furthermore, inactive mutants had a dominant negative effect, suggesting that the signaling module is a multimer. We constructed a chimeric molecule fusing the amino-terminal ectodomain of TLR3 to the transmembrane and carboxyl terminal domains of CD32a containing an immunoreceptor tyrosine-based motif. Expression of TLR3-CD32 in HEK293T cells and the myeloid cell line U937 resulted in surface localization of the receptor, whereas the nonrecombinant molecule was intracellularly localized. The synthetic double-stranded RNAs poly(I-C) and poly(A-U) induced calcium mobilization in a TLR3-CD32 stably transfected U937 clone but not in control cells transfected with other constructs. An anti-TLR3 antibody also induced Ca(2+) flux but only when cross-linked by a secondary anti-immunoglobulin antibody, confirming that multimerization by the ligand is a requirement for signaling. The inhibitors of lysosome maturation, bafilomycin and chloroquine, inhibited the poly(I-C)-induced biological response in immune cells, showing that TLR3 interacted with its ligand in acidic subcellular compartments. Furthermore, TLR3-CD32 activation with poly(I-C) was only observed within a narrow pH window (pH 5.7-6.7), whereas anti-TLR3-mediated Ca(2+) flux was pH-insensitive. The importance of an acidic pH for TLR3-ligand interaction becomes critical when using oligomeric poly(I-C) (15-40-mers). These observations demonstrate that engagement of TLR3 by poly(I-C) at an acidic pH, probably in early phagolysosomes or endosomes, induces receptor aggregation leading to signaling.

Amino Acid Sequence↗
PubMed1993

Influence of DNA base sequence on the binding energetics of actinomycin D.

The influences of base sequence on the thermodynamic properties associated with the interaction of actinomycin D with DNA are examined. It has been previously established that GpC steps of double-helical DNAs are highly preferred binding sites for actinomycin D. In this study, a series of oligonucleotides was designed and synthesized to probe the effects of flanking base sequence (adjacent to the GpC step) and novel non-GpC binding sites on the binding of actinomycin D. The use of these oligonucleotides provides a direct method for quantitating sequence specificities and actinomycin D binding energetics. Effects of different 5' and 3' flanking nucleotides on the interactions of actinomycin with the core GpC binding sites were examined using UV-visible spectrophotometric methods, and changes in binding energetics were quantitated. These studies demonstrate strong actinomycin D binding affinities to both classical GpC and an atypical non-GpC site. Enthalpy and entropy components of the DNA binding energetics for the GpC binding sites are compared and correlated with those determined for actinomycin D binding to the high-affinity non-GpC site of an 11-mer containing TGGGT as the central sequence. This TGGGT site, first suggested to be a high-affinity sequence in our earlier photoaffinity labeling studies, exhibits binding of actinomycin D comparable in strength to that of traditional actinomycin D binding sites (i.e., GpC steps). From these studies, the overall affinity and specific thermodynamic contributions (delta H degree, delta S degree) to binding of actinomycin D are demonstrated to be highly influenced both by the sequence at the intercalation site and by neighboring bases which flank the intercalation site.

Amino Acid Sequence↗
PubMed1997

Residues at the carboxy terminus of T4 DNA polymerase are important determinants for interaction with the polymerase accessory proteins.

Three T4 DNA polymerase accessory proteins (44P/62P and 45P) stimulate the polymerase (pol) activity and the 3'-5' exonuclease (exo) activity of T4 DNA polymerase (43P) on long, double-stranded DNA substrates. The 44P/62P "clamp loader" facilitates the binding of 45P, the "sliding clamp", to DNA that is primed for replication. Using a series of truncated 43P mutants, we identified a region at the extreme carboxy terminus of the DNA polymerase that is required for its interaction with accessory proteins. Truncation mutants of 43P lacking the carboxy-terminal 3, 6, or 11 residues retained full pol and exo activity on short synthetic primer-templates. However, the ability of the accessory proteins to enhance these activities on long double-stranded DNA templates was drastically reduced, and the extent of the reduction in activity was greater as more residues were deleted. One of the truncation mutants (N881), which had 17 residues removed from the carboxy terminus, showed reduced binding affinity and diminished pol activity but enhanced exo activity upon incubation with a small primer-template. The exo activity of the N881 mutant, on short, single-stranded DNA was unchanged, however, compared to the wild-type enzyme. These results are consistent with inferences drawn from the crystal structure of a DNA polymerase from a related T-even phage, RB69, where the carboxy-terminal 12 residues (equivalent to the 11 residues of 43P from phage T4) protrude from the thumb domain and are free to interact with complementary surfaces of the accessory proteins. The structural integrity of the thumb region in the N881 mutant is probably perturbed and could account for its reduced binding affinity and pol activity when incubated with short, double-stranded DNA substrates.

Amino Acid Sequence↗