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PubMed1994

Crystallization and low temperature diffraction studies of the DNA binding domain of the single-stranded DNA binding protein from Escherichia coli.

The DNA binding domain of the single-stranded DNA binding protein from Escherichia coli has been overproduced, purified and crystallized in a form suitable for X-ray diffraction studies. Crystals were produced by dialysis against low ionic strength buffer at high pH. The crystals belong to space group I222 or I2(1)2(1)2(1) with a = 82.47 A, b = 65.27 A and c = 46.50 A. Data were collected at several temperatures and a significant improvement in data quality was observed with a decrease in temperature. On occasion, with the decrease in temperature, a transformation to a primitive space group was observed and temperature appears to play a key role in this transformation. A complete native data set has been collected to 2.57 A at -15 degrees C.

Bacterial Proteins↗
PubMed2005

DNA-binding dependent and independent functions of WT1 protein during human hematopoiesis.

The Wilms tumor gene 1 (WT1) encodes a zinc-finger-containing transcription factor highly expressed in immature hematopoietic progenitor cells. Overexpression and presence of somatic mutations in acute leukemia indicate a role for WT1 in the pathogenesis of leukemia. CD34+ progenitor cells were transduced with one splice variant of human WT1 without the KTS insert in the zinc-finger domain, WT1(+/-), and with a deleted mutant of WT1 lacking the entire zinc-finger region, WT1(delZ), thus incapable of binding DNA. We show that inhibition of erythroid colony formation and differentiation is absolutely dependent on the DNA-binding zinc-finger domain of WT1. Unexpectedly, however, WT1(delZ) was equally effective as wild type protein in the reduction of myeloid clonogenic growth as well as in stimulation of myeloid differentiation, as judged by the expression of cell surface CD11b. Expression of neither WT1(+/-) nor WT1(delZ) upregulated mRNA for the cdk inhibitor p21(Waf1/Cip1) or p27Kip1. Our results demonstrate that WT1 affects proliferation and differentiation in erythroid and myeloid cells by different molecular mechanisms, and suggest that mutations affecting the zinc-finger domain of WT1 could interfere with normal differentiation in the pathogenesis of leukemia.

Antigens, CD34↗
PubMed1991

The DNA-binding domain of nuclear factor I is sufficient to cooperate with the adenovirus type 2 DNA-binding protein in viral DNA replication.

Recombinant baculoviruses have been constructed which express the full-length nuclear factor I (NFI) protein or a derivative of NFI that contains only the DNA-binding domain of the protein in infected insect cells. Both proteins were purified from insect cells infected with the respective baculoviruses and tested for their ability to cooperate with the adenovirus type 2 (Ad2) DNA-binding protein during virus replication. DNase I protection experiments demonstrated that the viral DNA-binding protein increased the affinity of both the full-length NFI and the DNA-binding domain of NFI for their recognition site in the Ad2 origin of DNA replication. As a consequence, the NFI-dependent increase in the efficiency of DNA replication observed upon addition of viral DNA-binding protein was the same when the full-length or DNA-binding domain derivative of NFI was added. Thus it appears that all of the activities associated with the ability of NFI to stimulate Ad2 DNA replication are located within the DNA-binding domain of the protein.

Adenoviridae↗
PubMed1999

NikR is a ribbon-helix-helix DNA-binding protein.

Escherichia coli NikR, a repressor with homologs in other bacteria and archaea, was identified as a potential new member of the ribbon-helix-helix (beta-alpha-alpha) family of transcription factors in profile based sequence searches and in structure prediction experiments. Biophysical and biochemical characterization of the N-terminal domain of NikR show that it has many features expected of a beta-alpha-alpha protein including alpha-helical content, dimeric solution form, concentration dependent thermal stability, and ability to bind DNA in sequence-specific manner. Mutation of a residue predicted to be important for DNA-binding reduces operator affinity but does not affect the secondary structure or stability of the protein.

Amino Acid Sequence↗
PubMedDate not supplied

Complex of simian virus large T antigen and p53 can bind DNA specifically.

The DNA-binding activity of p53 was suggested to be abrogated by binding of large T antigen. However we demonstrate that a fraction of p53 can form a complex with large T antigen and still bind DNA specifically. In EMSA analysis, both PAb 416 and 419 anti-large-T antibodies could induce supershift on p53-DNA complex using extracts derived from two T-antigen-transformed cell lines. In EMSA-Western analysis, one of the p53-complexes was shown to contain large T antigen.

Animals↗
PubMed1993

Immunization of BALB/c mice with a monoclonal anti-DNA antibody induces an anti-idiotypic antibody reactive with a cell-surface DNA binding protein.

DNA binds to cell-surface proteins on human and murine leukocytes and induces secretion of the cytokine interleukin 6 (IL-6). Cell-surface DNA binding molecules have been shown to serve as target antigens for the production of autoantibodies in patients with systemic lupus erythematosus (SLE), and in lupus-prone mice. Recent studies have demonstrated that a subset of anti-anti-DNA antibodies, isolated from patients with SLE, are idiotypically related to antibodies reactive with a cell-surface DNA binding molecule. We now report that immunization of normal mice with a murine monoclonal anti-DNA antibody induces an anti-idiotypic response which has reactivity with a cell-surface DNA binding molecule. An anti-idiotypic anti-DNA monoclonal antibody (LB17) was isolated from the spleen of an immunized mouse. This monoclonal antibody blocked the binding of DNA to murine splenocytes and mimicked the functional effect of DNA by stimulating the secretion of IL-6. These experiments provide further evidence for an idiotypic connectivity between antibodies to cell-surface DNA binding proteins and anti-DNA antibodies. It is hypothesized that this idiotypic system is part of the network of natural autoantibodies and that its perturbation may give rise to pathogenic antibodies.

Animals↗
PubMed1983

Effects of the adenovirus H5ts125 and H5ts107 DNA binding proteins on DNA replication in vitro.

Genetic and biochemical studies of adenovirus (Ad) DNA synthesis in vitro demonstrate that the Ad DNA binding protein (Ad DBP) is not necessary for the initiation of Ad DNA synthesis but is required for chain elongation. The DBP, which enhances early elongation to the 26th deoxynucleotide by approximately two- to fourfold, is absolutely required as chain elongation proceeds further. Ad DNA synthesis was assayed in a system requiring Ad DNA covalently linked at each 5' terminus to a protein (Ad DNA-pro), various fractions of Ad-infected cytoplasm, and an extract of uninfected Hela nuclei. Initiation of Ad DNA replication was measured by the formation of a covalent complex between the 80,000 dalton preterminal protein (pTP) and 5' dCMP. DNA binding proteins from two ts mutants, H5ts125 and H5ts107, have been purified and shown to be functional at 30 degrees but inactive at 38 degrees in an in vitro elongation system dependent on purified proteins. Chymotryptic cleavage of the 72K wild-type Ad2 DBP produces a 34K carboxyl terminal fragment which retains full activity in the in vitro elongation of Ad DNA.

Adenoviruses, Human↗
PubMed2004

The development and applications of nonradioactive plate-formatted DNA-binding assay for Ku70/80, a multifunctional DNA-binding protein complex.

Ku is a heterodimer composed of p70 and p80, and is the regulatory subunit of DNA-dependent protein kinase. As a multifunctional DNA-binding protein complex, Ku plays important roles in DNA damage repair through non-homologous end joining and in V(D)J recombination. In addition, Ku has also been implicated in various biological functions including growth control, cell proliferation, cell cycle, chromosome maintenance, transcriptional regulation, apoptosis, and viral infection. In particular, using our Inverse Genomics (Immusol, Inc., San Diego, CA) platform technology, we recently identified Ku80 as an essential co-factor for human immunodeficiency virus replication. Although Ku has been studied extensively in the past years, its in-depth study as well as development as a drug target has been limited by conventional DNA-binding activity assay. Here we describe the development and applications of a nonradioactive DNA binding assay in the 96-well format. We show that this plate-formatted assay is more sensitive and allows for direct quantification when compared with an electrophoretic mobility shift assay. The establishment of this assay will not only facilitate structure and function studies on Ku, but also help the development of Ku protein or its DNA repair enzyme complex as a drug target.

Animals↗
PubMed1993

I kappa B alpha-mediated inhibition of v-Rel DNA binding requires direct interaction with the RXXRXRXXC Rel/kappa B DNA-binding motif.

Rel family proteins bind to kappa B DNA sites, form heterodimers with one another, and modulate expression of genes linked to kappa B motifs. I kappa B factors associate with Rel proteins, inhibit Rel DNA binding in vitro, and displace DNA from DNA-bound Rel complexes. We have investigated the mechanism by which the p40/I kappa B alpha inhibitor interfers with Rel DNA-binding activity. Here, we report that p40 contacts the RXXRXRXXC DNA-binding motif conserved in all Rel family proteins, in addition to associating with the nuclear localizing sequence. Competition assays with a Rel-derived peptide comprising the DNA-binding region specifically alleviated p40-mediated inhibition of v-Rel DNA-binding activity, whereas a covalently modified Rel peptide was inactive. Combined, these results indicate that I kappa B alpha interaction with the RXXRXRXXC motif is required for inhibition of v-Rel DNA binding and suggest that nuclear I kappa B factors may be critical for regulating transcription by Rel family proteins.

Amino Acid Sequence↗
PubMed1990

Characterization of a major DNA-binding domain in the herpes simplex virus type 1 DNA-binding protein (ICP8).

We have studied the major DNA-binding protein (ICP8) from herpes simplex virus type 1 to identify its DNA-binding site. Since we obtained our protein from a cell line carrying multiple chromosomally located copies of the ICP8 gene, we first analyzed this protein to assess its similarity to the corresponding viral protein. Our protein resembled the viral protein by molecular weight, response to antibody, preference for binding single-stranded DNA, and ability to lower the melting temperature of poly(dA-dT). To define the DNA-binding domain, we subjected the protein to limited trypsin digestion and separated the peptide products on a sodium dodecyl sulfate-polyacrylamide gel. These fragments were then transferred to a nitrocellulose membrane, renatured in situ, and tested for their ability to bind DNA. From this assay, we identified four fragments which both bound DNA and exhibited the expected binding preference for single-stranded DNA. The sequence of the smallest of these fragments was determined and corresponds to a polypeptide spanning residues 300 to 849 in the intact protein. This peptide contains several regions which may be important for DNA binding based on sequence similarities in single-stranded DNA-binding proteins from other herpesviruses and, in one case, on a conserved sequence found in more distant procaryotic and eucaryotic proteins.

Amino Acid Sequence↗
PubMed1992

Determination of recognition-sequences for DNA-binding proteins by a polymerase chain reaction assisted binding site selection method (BSS) using nitrocellulose immobilized DNA binding protein.

We have developed a simple procedure for rapid determination of a DNA sequence recognized by a DNA binding protein based on immobilization of the protein on nitrocellulose filters. The procedure consists of the following steps: A recombinant protein with a functional DNA binding domain is expressed in E. coli. The protein is purified to homogeneity, immobilized on nitrocellulose paper, and exposed to a pool of double stranded oligonucleotides carrying in the central part a 20 bp random sequence, which is flanked by conserved sequences with restriction endonuclease recognition sites for analytical and subcloning purposes and sequences complementary to polymerase chain reaction primers. Oligonucleotides retained by the DNA-binding protein are liberated by increasing the ionic strength and used in a new binding process after amplification by the polymerase chain reaction technique. Finally the amplified product is cloned for determination of the DNA sequence selected by the DNA-binding protein. Murine Zn-finger and basic helix-loop-helix DNA binding proteins were used to demonstrate the efficiency of the method. We show that the yield of oligonucleotides binding to the protein was increased by several consecutive rounds of filter binding and amplification, and that the protein extracted a specific sequence from the pool of random oligonucleotides.

Animals↗
PubMed2000

p53 C-terminal interaction with DNA ends and gaps has opposing effect on specific DNA binding by the core.

In addition to binding DNA in a sequence-specific manner, the p53 tumour suppressor protein can interact with damaged DNA. In order to understand which structural features in DNA the C-teminal domain recognises we have studied the interaction of p53 protein with different types of DNA oligonucleotides imitating damaged DNA. Here we show that one unpaired nucleotide within double-stranded (ds)DNA is sufficient for recognition by the p53 C-terminus, either as a protruding end or as an internal gap in dsDNA. C-terminal interaction with DNA ends facilitated core domain binding to DNA, whereas interaction with gaps prevented core domain-DNA complexing, implying that p53 might adopt distinct conformations upon binding to different DNA lesions. These observations suggest that both single-strand and double-strand breaks can serve as a target for p53 C-terminal recognition in vivo and indicate that p53 might recruit different repair factors to the sites of damaged DNA depending on the type of lesion.

Base Pairing↗
PubMed1988

Sulfhydryl group content of chicken progesterone receptor: effect of oxidation on DNA binding activity.

DNA binding activity of chicken progesterone receptor B form (PRB) and A form (PRA) has been examined. This activity is strongly dependent upon the presence of thiols in the buffer. Stability studies showed that PRB was more sensitive to oxidation than was PRA. Receptor preparations were fractionated by DNA-cellulose chromatography to DNA-positive and DNA-negative subpopulations, and sulfhydryl groups were quantified on immunopurified receptor by labeling with [3H]-N-ethylmaleimide. Labeling of DNA-negative receptors with [3H]-N-ethylmaleimide showed 21-23 sulfhydryl groups on either PRA or PRB form when the proteins were reduced and denatured. A similar number was seen without reduction if denatured DNA-positive receptor species were tested. In contrast, the DNA-negative PRB had only 10-12 sulfhydryl groups detectable without reduction. A similar number (12-13 sulfhydryl groups) was found for PRA species that lost DNA binding activity after exposure to a nonreducing environment in vitro. We concluded that the naturally occurring receptor forms unable to bind to DNA, as well as receptor forms that have lost DNA binding activity due to exposure to a nonreducing environment in vitro, contain 10-12 oxidized cysteine residues, likely present as disulfide bonds. Since we were unable to reduce the disulfide bonds when the native DNA-negative receptor proteins were treated with dithiothreitol (DTT), we speculate that irreversible loss of DNA binding activity of receptor in vitro is due to oxidation of cysteine residues that are not accessible to DTT in the native state.

Animals↗
PubMed1994

Characterization of baculovirus recombinant wild-type p53. Dimerization of p53 is required for high-affinity DNA binding and cysteine oxidation inhibits p53 DNA binding.

A high-yield, rapid and non-denaturing purification protocol for baculovirus recombinant wild-type p53 is described. Gel-filtration chromatography and chemical cross-linking experiments indicated that purified p53 assembles into multimeric forms ranging from tetramer to higher oligomers. A gel-mobility-shift assay and protein-DNA cross-linking studies demonstrated that purified baculovirus recombinant p53 binds to consensus DNA target as a dimer but that additional p53 molecules may then associate with the preformed p53-dimer-DNA complexes to form larger p53 DNA complexes. These observations suggest that the p53 tetramers and higher oligomers that form the minimal p53 association in solution dissociate upon DNA binding to form p53 dimer-DNA complexes. Binding of the mAB PAb 421 to the oligomerization-promoting domain on p53 stimulated sequentially formation of both p53-dimer-DNA and larger p53-DNA complexes. This observation suggests that factors may exist in vivo that could participate in the formation and the stabilization of the various p53-DNA complexes. Further characterization of the purified p53 revealed that the protein possesses highly reactive cysteine residues. We show that intrachain disulfide bonds form within the purified p53 molecules during storage in the absence of reducing agent. Zn2+ binding to p53 protect sulfhydryl groups from oxidation. Cysteine oxidation by intramolecular disulfide-bond formation did not modify the wild-type immunoreactive phenotype of the p53 protein but totally inhibited its DNA-binding activities. The oxidation of the p53 cysteine residues was also observed for nuclear p53 in baculovirus-infected insect cells. The redox status of the nuclear p53 regulates its DNA-binding activity in vitro confirming the essential role of the reduced state of cysteine residues in p53 for detectable DNA-binding activity.

Animals↗
PubMed2005

The redox state of the baculovirus single-stranded DNA-binding protein LEF-3 regulates its DNA binding, unwinding, and annealing activities.

The single-stranded (ss) DNA-binding protein LEF-3 of Autographa californica multinucleocapsid nucleopolyhedrovirus promoted Mg(2+)-independent unwinding of DNA duplexes and annealing of complementary DNA strands. The unwinding and annealing activities of LEF-3 appeared to act in a competitive manner and were determined by the ratio of protein to DNA. At subsaturating and saturating concentrations, LEF-3 promoted annealing, whereas it promoted unwinding at oversaturation of DNA substrates. The LEF-3 binding to ssDNA and unwinding activity were sensitive to redox agents and were inhibited by oxidation of thiol groups in LEF-3 with 1,1'-azobis(N,N-dimethylformamide) (diamide) or by modification with the thiol-conjugating agent N-ethylmaleimide. Both oxidation and alkylation increased the dissociation constant of the interaction with model oligonucleotides indicating a decrease in an intrinsic affinity of LEF-3 for ssDNA. These results proved that free thiol groups are essential both for LEF-3 interaction with ssDNA and for DNA unwinding. In contrast, oxidation or modification of thiol groups stimulated the annealing activity of LEF-3 partially due to suppression of its unwinding activity. Treatment of LEF-3 with the reducing agent dithiothreitol inhibited annealing, indicating association of this activity with the oxidized protein. Thus, the balance between annealing and unwinding activities of LEF-3 was determined by the redox state of protein with the oxidized state favoring annealing and the reduced state favoring unwinding. An LEF-3 mutant in which the conservative cysteine Cys(214) was replaced with serine showed both a decreased binding to DNA and a reduced unwinding activity, thus indicating that this residue might participate in the regulation of LEF-3 activities.

Animals↗
PubMed2004

Sensing of remote oxyanion binding at the DNA binding domain of the molybdate-dependent transcriptional regulator, ModE.

The molybdate-dependent transcriptional regulator ModE of Escherichia coli displays a large (50%) quenching of its intrinsic tryptophan fluorescence on binding molybdate. The changes in fluorescence have been exploited to analyse the binding of molybdate to ModE. Utilising site-directed mutagenesis, a series of phenylalanine substitutions for the three tryptophans of ModE (Trp49, Trp131 and Trp186) have been constructed, to yield three mono-Trp-containing derivatives. This has allowed an assessment to be made of the contribution of each of the three tryptophans to the spectral changes observed on binding molybdate; these are most distinctive for Trp186. Linkage between the DNA-binding and molybdate-binding sites (some 55 angstroms apart) is shown by (a) the small, but definite, effect of molybdate on the fluorescence of Trp49 which is located at the DNA-binding winged helix-turn-helix domain, and (b) the finding that the binding of either ligand is enhanced in the presence of the other. The studies demonstrate that the mono-Trp derivatives of ModE could be useful tools with which to study the signal transduction processes specifically associated with molybdate-dependent transcriptional regulation and that this approach may have wider implications for analysis of other regulated systems.

Amino Acid Sequence↗
PubMed1998

Thermodynamics of specific and non-specific DNA binding by the c-Myb DNA-binding domain.

The thermodynamics of the c-Myb DNA-binding domain (R2R3) interaction with its target DNA have been analyzed using isothermal titration calorimetry and amino acid mutagenesis. The enthalpy of association between the standard R2R3, the Cys130 mutant substituted with Ile, and the cognate DNA is -12.5 (+/- 0.1) kcal mol-1 at pH 7.5 and at 20 degrees C, and this interaction is enthalpically driven throughout the physiological temperature range. In order to understand the DNA recognition mechanism, several pairs of interactions were investigated using single and multiple-base alterations with single and multiple-amino acid substituted mutants. The interactions between the standard R2R3 and many non-cognate DNAs were accompanied by binding enthalpy changes and heat capacity changes, although their affinities were reduced. The roles of the electrostatic interactions in binding to the cognate and the non-cognate DNAs were also analyzed from the dependency of the thermodynamic parameters on the salt concentration. The heat capacity change was found to be significantly dependent upon the salt concentration. Several mutant proteins bound to the multiple-base altered DNA with very small enthalpy changes, although they bound to the cognate and the single-base altered DNAs with detectable enthalpy and heat capacity changes. From the thermodynamic cycles derived from the DNA binding of the amino acid substituted R2R3 to the base substituted DNA duplexes, the individual thermodynamic mechanisms of the specific DNA recognition of R2R3 were dissected. The local folding mechanism was highlighted by the substitution of Pro with either Gly or Ala at the linker between R2 and R3. The characteristic thermodynamic features of specific and non-specific DNA binding are discussed.

Amino Acid Substitution↗