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At least 217 records · Page 12Linked to original sources

PubMed1993

Pleiotropic effect of the human T-cell leukemia virus Tax protein on the DNA binding activity of eukaryotic transcription factors.

The Tax protein, encoded by the human T-cell leukemia virus type I, is a potent activator of viral and cellular gene transcription. Tax does not bind DNA directly but appears to trans-activate through an interaction with host-cell transcription factors that recognize sequences within the promoters of Tax-responsive genes. Cellular transcriptional activators implicated in mediating Tax trans-activation include members of the activating transcription factor/cAMP response element binding protein (ATF/CREB) family of proteins, serum response factor, Fos-Jun, and NF-kappa B. Recent evidence suggests that Tax may stimulate human T-cell leukemia virus type I transcription, at least in part, through enhanced binding of ATF/CREB proteins to their recognition elements within the Tax-responsive 21-bp repeats of the viral promoter. In this report, we demonstrate that Tax also enhances the site-specific DNA binding activity of serum response factor and Fos-Jun and modestly enhances the binding of the NF-kappa B subunits, p50 and p65. We also show that Tax increases the DNA binding activity of the eukaryotic transcription factors ATF-1, Sp1, and GAL4. These results are consistent with the finding that Tax is highly pleiotropic and suggest that Tax trans-activation may involve enhancement in the DNA binding activity of target transcriptional regulatory proteins. In addition, we show that the mechanism of Tax-enhanced DNA binding activity does not involve an alteration in the redox state of the target protein.

Base Sequence↗

PubMed1992

elk-1 domains responsible for autonomous DNA binding, SRE:SRF interaction and negative regulation of DNA binding.

The ets oncogene superfamily consists of a family of sequence-specific DNA-binding transcriptional activator proteins. We have previously identified, cloned and characterized one of the divergent ets-related members elk-1 and shown that it codes for a sequence-specific DNA-binding transcriptional activator. We have also shown that elk-1 forms SRF (Serum Response Factor) dependent ternary complex with SRE (Serum Response Element), similar to p62TCF. In this report, we have mapped the DNA-binding domain of the elk-1 protein (EDB, elk-1 DNA Binding domain) to the 76 amino acid ets homology region. We have also mapped the SRF interaction domain of the elk-1 protein (ESI, elk-1 SRF Interaction domain) to the carboxy-terminal region of the EDB domain. Ternary complex formation by elk-1 requires both EDB and ESI domains of the elk-1 protein. Our results also show that the EDB domain of the elk-1 protein (residues 1-89) binds SRE autonomously, unlike full-length elk-1 protein, suggesting the presence of a potential Negative Regulatory DNA binding domain (NRD) which prevents the binding of elk-1 protein to SRE. Interaction of SRF with the ESI domain allows the elk-1 protein to bind to SRE. Thus elk-1 belongs to a class of transcriptional factors that are involved in gene regulation not only by autonomous DNA binding but also by indirect DNA binding through recruitment by cellular factors.

DNA↗

PubMed1995

HhaI and HpaII DNA methyltransferases bind DNA mismatches, methylate uracil and block DNA repair.

The hydrolytic deamination of 5-methylcytosine (5-mC) to thymine (T) is believed to be responsible for the high mutability of the CpG dinucleotide in DNA. We have shown a possible alternate mechanism for mutagenesis at CpG in which HpaII DNA-(cytosine-5) methyltransferase (M.HpaII) can enzymatically deaminate cytosine (C) to uracil (U) in DNA [Shen, J.-C., Rideout, W.M., III and Jones, P.A., Cell, 71, 1073-1080, (1992)]. Both the hydrolytic deamination of 5-mC and enzymatic deamination of C create premutagenic DNA mismatches (G:U and G:T) with the guanine (G) originally paired to the normal C. Surprisingly, we found that DNA-(cytosine-5) methyltransferases have higher affinities for these DNA mismatches than for their normal G:C targets and are capable of transferring a methyl group to the 5-position of U, creating T at low efficiencies. This binding by methyltransferase to mismatches at the recognition site prevented repair of G:U mismatches by uracil DNA glycosylase in vitro.

Base Sequence↗

PubMed1997

Proposed mechanism for the stabilization of nuclear receptor DNA binding via protein dimerization.

Hepatocyte nuclear factor 4 (HNF-4) defines a new subgroup of nuclear receptors that exist in solution and bind DNA exclusively as homodimers. We recently showed that the putative ligand binding domain (LBD) of HNF-4 is responsible for dimerization in solution and prevents heterodimerization with other receptors. In this report, the role of the LBD in DNA binding by HNF-4 is further investigated by using electrophoretic mobility shift analysis. A comparison of constructs containing either the DNA binding domain (DBD) alone or the DBD plus the LBD of HNF-4 showed that dimerization via the DBD was sufficient to provide nearly the full DNA binding affinity of the full-length HNF-4. In contrast, dimerization via the DBD was not sufficient to produce a stable protein-DNA complex, whereas dimerization via the LBD increased the half-life of the complex by at least 100-fold. Circular permutation analysis showed that full-length HNF-4 bent DNA by approximately 80 degrees while the DBD bent DNA by only 24 degrees. Nonetheless, analysis of other constructs indicated that the increase in stability afforded by the LBD could be explained only partially by an increased ability to bend DNA. Coimmunoprecipitation studies, on the other hand, showed that dimerization via the LBD produced a protein-protein complex that was much more stable than the corresponding protein-DNA complex. These results led us to propose a model in which dimerization via the LBD stabilizes the receptor on DNA by converting an energetically favorable two-step dissociation event into an energetically unfavorable single-step event. Implications of this one-step model for other nuclear receptors are discussed.

Binding Sites↗

PubMed1989

DNA binding property of vitamin D3 receptors associated with 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3.

Using [3H]-26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3 (F6-1,25-(OH)2D3), we have examined its ability to bind to the 1,25-(OH)2D3 receptor, and the ability of the resulting complex to bind DNA. The binding sites for [3H]F6-1,25-(OH)2D3 in the chick intestinal receptor represented a limited number of saturable sites for which 1,25-(OH)2D3 competes. 1,25-Dihydroxyvitamin D3 is three times more active than F6-1,25-(OH)2D3 in displacing [3H]F6-1,25-(OH)2D3. By affinity chromatography using DNA-Sephadex, the [3H]F6-1,25-(OH)2D3 receptor complex eluted from the column in a single peak at 0.14 M KCl, while [3H]-1,25-(OH)2D3 receptor complex eluted at 0.13 M KCl. These results indicate that F6-1,25-(OH)2D3 and 1,25-(OH)2D3 recognize the same binding site of the receptor and that the F6-1,25-(OH)2D3 receptor complex binds DNA more tightly than the 1,25-(OH)2D3 receptor complex. We suggest that the higher binding affinity for DNA may contribute to the greater biological activity of F6-1,25-(OH)2D3.

Animals↗

PubMed1998

Identification of an additional negative regulatory region for p53 sequence-specific DNA binding.

The DNA binding activity of p53 is crucial for its tumor suppressor function and is subject to tight regulation. Previous studies revealed that the inhibitory function of the p53 C terminus is implicated in the latent, low affinity sequence-specific DNA binding activity of p53 in the uninduced state. Sequence-specific DNA binding of p53 has been shown to be activated by several posttranslational modifications and interacting proteins that target predominantly the C terminus. Moreover, several authors have shown that synthetic peptides corresponding to p53 C-terminal sequences activate p53 sequence-specific DNA binding. In an effort to identify the interaction site of p53 with these activating peptides we assessed complex formation between p53 deletion constructs and C-terminal activating peptides by peptide affinity precipitation. This study revealed that two distal regions of the p53 molecule contribute synergistically to the interaction with activating C-terminal peptides: amino acids 80-93 and 364-393. The C-terminal residues 364-393 are already well characterized as having negative regulatory function. DNA binding analyses with these deletion constructs reveal a comparable negative regulatory activity for residues 80-93, defining this region as a previously unidentified negative regulatory domain of p53. Furthermore, synthetic peptides spanning this newly identified proline-rich negative regulatory region (residues 80-93) are able to activate p53 sequence-specific DNA binding in vitro. We suggest that both negative regulatory regions, residues 80-93 and 364-393, contribute cooperatively to the maintenance of the latent, low-affinity DNA binding conformation of p53.

Binding Sites↗

PubMed1991

Inefficient homooligomerization contributes to the dependence of myogenin on E2A products for efficient DNA binding.

Myogenin is a muscle-specific transcription factor that can activate myogenesis; it belongs to a family of transcription factors that share homology within a basic region and an adjacent helix-loop-helix (HLH) motif. Although myogenin alone binds DNA inefficiently, in the presence of the widely expressed HLH proteins E12 and E47 (encoded by the E2A gene), it forms heterooligomers that bind with high affinity to a DNA sequence known as a kappa E-2 site. In contrast, E47 and to a lesser extent E12 are both able to bind the kappa E-2 site relatively efficiently as homooligomers. To define the relative contributions of the basic regions of myogenin and E12 to DNA binding and muscle-specific gene activation, we created chimeras of the two proteins by swapping their basic regions. We showed that myogenin's weak affinity for the kappa E-2 site is attributable to inefficient homooligomerization and that the myogenin basic domain alone can mediate high-affinity DNA binding when placed in E12. Within a heterooligomeric complex, two basic regions were required to form a high-affinity DNA-binding domain. Basic-domain mutants of myogenin or E2A gene products that cannot bind DNA retained the ability to oligomerize and could abolish DNA binding of the wild-type proteins in vitro. These myogenin and E2A mutants also acted as trans-dominant inhibitors of muscle-specific gene activation in vivo. These findings support the notion that muscle-specific gene activation requires oligomerization between myogenin and E2A gene products and that E2A gene products play an important role in myogenesis by enhancing the DNA-binding activity of myogenin, as well as other myogenic HLH proteins.

Amino Acid Sequence↗

PubMed2006

The DNA-binding domain of yeast Hsf1 regulates both DNA-binding and transcriptional activities.

The heat shock transcription factor (HSF) is a key regulator of the heat shock response. In Saccharomyces cerevisiae, the transcription activating ability of Hsf1 is repressed by its DNA-binding domain, but the detailed mechanism by which the inhibitory function is relieved in response to stress remains unknown. In this study, we isolated and characterized three hsf1 mutants with temperature-sensitive mutations in the DNA-binding domain. Two mutations inhibited DNA-binding activity, leading to decreased expression of target genes. The third mutation caused transcriptional defects without affecting DNA binding, and its suppressor mutation was located in a region important for sensing heat shock. These results indicate that the DNA-binding domain regulates both the DNA-binding and transcriptional activities of Hsf1, and suggest that these functions are located within discrete regions of the DNA-binding domain.

Amino Acid Substitution↗

PubMed1989

Levels of DNA topoisomerases, single-stranded-DNA-binding protein, and DNA polymerase I in rho+ and rho-15 strains of Escherichia coli.

The Escherichia coli rho-15 mutant, which is highly defective in transcription termination, was examined to see whether its reduced DNA superhelicity could be explained by altered expression of proteins that may affect DNA structure. Levels of DNA gyrase and topoisomerase I were normal; levels of single-stranded-DNA-binding protein, DNA polymerase I, and a protein tentatively identified as Lon were significantly altered.

DNA Polymerase I↗

PubMed1988

Helix lap-joints as ion-binding sites: DNA-binding motifs and Ca-binding "EF hands" are related by charge and sequence reversal.

The DNA-binding helix pairs in gene repressor and activator proteins were compared with other approximately perpendicular pairs of adjacent helices in the known protein structures. Two other examples of closely matching conformations were found in cytochrome c peroxidase (residues 153-174) and in ribosomal L7/L12 protein (residues 68-89). Another group of such offset "lap-joints" are the Ca-binding "EF hand" structures, which bind a positive rather than a negative ligand. The EF hands turn out to match the DNA-binding motifs quite well (outside of the loop) if their sequence direction is reversed. This conformation is thus not as unusual as had been thought, but may have a more generalized role in ion binding and occasionally occur in a purely structural role.

Amino Acid Sequence↗

PubMed2003

Accessibility of nuclear chromatin by DNA binding polyamides.

Pyrrole-imidazole polyamides bind DNA with affinities comparable to those of transcriptional regulatory proteins and inhibit the DNA binding activities of components of the transcription apparatus. If polyamides are to be useful for the regulation of gene expression in cell culture experiments, one pivotal issue is accessibility of specific sites in nuclear chromatin. We first determined the kinetics of uptake and subcellular distribution of polyamides in lymphoid and myeloid cells using fluorescent polyamide-bodipy conjugates and deconvolution microscopy. Then cells were incubated with a polyamide-chlorambucil conjugate, and the sites of specific DNA cleavage in the nuclear chromatin were assayed by ligation-mediated PCR. In addition, DNA microarray analysis revealed that two different polyamides generated distinct transcription profiles. Remarkably, the polyamides affected only a limited number of genes.

Alkylation↗

PubMed1989

A type II DNA-binding protein genetically engineered for fluorescence spectroscopy: the "arm" of transcription factor 1 binds in the DNA grooves.

We examined the fluorescence properties of a mutant TF1 protein (transcription factor 1; a member of the type II class of DNA-binding proteins, DBPII) containing tryptophan in place of phenylalanine (TF1-W61) at position 61 in the "arms" of the protein dimer. The time-resolved fluorescence (excited at 295 nm) of Trp61 decays as a double exponential with lifetimes and amplitudes that are comparable to those found in other tryptophan-containing proteins and peptides, and the time-resolved fluorescence polarization decay indicates that the tryptophan residue possesses considerable internal flexibility, in agreement with crystal studies of the homologous HU protein. The tryptophan emission is quenched when TF1-W61 binds to DNA, and equilibrium studies based on fluorescence show that the nonspecific binding affinity of the TF1-W61 mutant to DNA is similar to that of wild-type TF1. Comparison of the time-resolved fluorescence decay and steady-state fluorescence intensity reveals at least two general classes of Trp61 in the DNA complexes. One class of tryptophans is partially quenched, and the extent of quenching in the complexes with various natural DNAs and synthetic double-stranded polynucleotides correlates with the spectral overlap between tryptophan emission and DNA absorption, indicating that through-space excitation energy transfer contributes to the observed quenching. Comparisons between experimentally determined energy transfer rates and model calculations suggest that the Trp61 is located in one of the DNA grooves at a distance of less than 7.5 A from the DNA helix axis. The second class of Trp61 is "totally" quenched, and we attribute this to tryptophan residues that are in direct contact with the DNA bases.(ABSTRACT TRUNCATED AT 250 WORDS)

Codon↗

PubMed1991

DNA binding specificity of mutant glucocorticoid receptor DNA-binding domains.

Mutation of a small number of amino acids in the DNA-binding domain of the estrogen receptor to the corresponding sequence of the glucocorticoid receptor switches the specificity of the receptor in transactivation assays (Mader, S., Kumar, V., de Verneuil, H., and Chambon, P. (1989) Nature 338, 271-274). We have made the corresponding reciprocal mutations in the context of the glucocorticoid receptor DNA-binding domain and studied the binding of wild type and mutant purified proteins to palindromic glucocorticoid and estrogen response elements as well as to elements of intermediate sequence, using gel mobility shift assays. We show here that a protein with two altered amino acids binds glucocorticoid and estrogen response elements with a low but equal affinity, whereas a protein with an additional changed residue has a high affinity for estrogen response elements but still retains a considerable affinity for glucocorticoid response elements. Using binding sites of intermediate sequence we have further characterized the interaction with DNA. The in vitro DNA binding results are confirmed by in vivo transactivation assays in yeast. Finally we suggest a testable model for amino acid/base pair interactions involved in recognition by the glucocorticoid receptor DNA-binding domain of its target sequence.

Amino Acid Sequence↗

PubMed2005

The bldC developmental locus of Streptomyces coelicolor encodes a member of a family of small DNA-binding proteins related to the DNA-binding domains of the MerR family.

The bldC locus, required for formation of aerial hyphae in Streptomyces coelicolor, was localized by map-based cloning to the overlap between cosmids D17 and D25 of a minimal ordered library. Subcloning and sequencing showed that bldC encodes a member of a previously unrecognized family of small (58- to 78-residue) DNA-binding proteins, related to the DNA-binding domains of the MerR family of transcriptional activators. BldC family members are found in a wide range of gram-positive and gram-negative bacteria. Constructed DeltabldC mutants were defective in differentiation and antibiotic production. They failed to form an aerial mycelium on minimal medium and showed severe delays in aerial mycelium formation on rich medium. In addition, they failed to produce the polyketide antibiotic actinorhodin, and bldC was shown to be required for normal and sustained transcription of the pathway-specific activator gene actII-orf4. Although DeltabldC mutants produced the tripyrrole antibiotic undecylprodigiosin, transcripts of the pathway-specific activator gene (redD) were reduced to almost undetectable levels after 48 h in the bldC mutant, in contrast to the bldC+ parent strain in which redD transcription continued during aerial mycelium formation and sporulation. This suggests that bldC may be required for maintenance of redD transcription during differentiation. bldC is expressed from a single promoter. S1 nuclease protection assays and immunoblotting showed that bldC is constitutively expressed and that transcription of bldC does not depend on any of the other known bld genes. The bldC18 mutation that originally defined the locus causes a Y49C substitution that results in instability of the protein.

Amino Acid Substitution↗

PubMed1993

The truncation that generated the v-cbl oncogene reveals an ability for nuclear transport, DNA binding and acute transformation.

The v-cbl oncogene is the transforming gene of the murine Cas NS-1 retrovirus which induces pre-B cell lymphomas and myeloid leukaemias. Sequencing of c-cbl has revealed that v-cbl was generated by a large truncation that removed 60% of the C-terminus of the corresponding protein. In this study we prepared antibodies to cbl and found that c-cbl encodes a 120 kDa protein which is localized in the cytoplasm with a cytosolic and cytoskeletal distribution. Immunofluorescence studies show a striking pattern of brightly staining vesicles in mitotic cells similar to that observed with cytokeratin antibodies. In contrast to p120c-cbl, which is exclusively cytoplasmic, the p100gag-v-cbl encoded by Cas NS-1 is localized in both the cytoplasm and the nucleus. This redistribution to the nucleus correlates with the ability of cbl to induce acute transformation. Furthermore the truncated protein encoded by v-cbl can bind DNA, unlike the full-length protein. These results suggest that the C-terminus of cbl is involved in the retention of p120c-cbl in the cytoplasm and the inhibition of DNA binding. The findings also suggest that a truncated protein encoded by c-cbl exists in the nucleus of normal cells.

3T3 Cells↗

PubMed2003

Drosophila DSP1 and rat HMGB1 have equivalent DNA binding properties and share a similar secondary fold.

The protein DSP1 belongs to the group of HMG-box proteins, which share the common structural feature of the HMG-box. This approximately 80 amino acid long motif binds DNA via the minor groove. DSP1 was discovered as a transcriptional co-repressor of Dorsal in Drosophila melanogaster and then was shown to participate to the remodeling of chromatin. By means of sequence alignment and gene organization, DSP1 was classified as the fly homologue of the vertebrate proteins HMGB1/2. DSP1 contains two HMG boxes flanked by two glutamine-rich domains at the N-terminus. In addition, the HMG domain of DSP1 displays two differences in its primary sequence as compared to the vertebrate HMGB1: a shorter acidic tail and a linker between the two boxes longer by 6 amino acids. By comparing several functional parameters of DSP1 with those of HMGB1, the present study establishes the functional equivalence of both proteins in terms of DNA recognition. The major structural difference between the two proteins, the glutamine-rich N-terminal tail of DSP1, which does not exist in HMGB1, did not interfere with any of the studied DNA-binding properties of the proteins.

Animals↗

PubMed1995

Phosphorylation of the Drosophila engrailed protein at a site outside its homeodomain enhances DNA binding.

The engrailed gene encodes a homeodomain-containing phosphoprotein that binds DNA. Here, we show that engrailed protein is posttranslationally modified in embryos and in embryo-derived cultured cells but is essentially unmodified when expressed in Escherichia coli. Engrailed protein produced by bacteria can be phosphorylated in nuclear extracts prepared from Drosophila embryos, and phosphotryptic peptides from this modified protein partly reproduce two-dimensional maps of phosphotryptic fragments obtained from metabolically labeled engrailed protein. The primary embryonic protein kinase modifying engrailed protein is casein kinase II (CK-II). Analysis of mutant proteins revealed that the in vitro phosphoacceptors are mainly clustered in a region outside the engrailed homeodomain and identified serines 394, 397, 401, and 402 as the targets for CK-II phosphorylation. CK-II-dependent phosphorylation of an N-truncated derivative of engrailed protein purified from bacteria increased its DNA binding 2-4-fold.

Amino Acid Sequence↗

PubMed2001

DNA-binding specificity and dimerization of the DNA-binding domain of the PEND protein in the chloroplast envelope membrane.

The PEND protein is a DNA-binding protein in the inner envelope membrane of a developing chloroplast, which may anchor chloroplast nucleoids. Here we report the DNA-binding characteristics of the N-terminal basic region plus leucine zipper (bZIP)-like domain of the PEND protein that we call cbZIP domain. The basic region of the cbZIP domain diverges significantly from the basic region of known bZIP proteins that contain a bipartite nuclear localization signal. However, the cbZIP domain has the ability to dimerize in vitro. Selection of binding sites from a random sequence pool indicated that the cbZIP domain preferentially binds to a canonical sequence, TAAGAAGT. The binding site was also confirmed by gel mobility shift analysis using a representative binding site within the chloroplast DNA. These results suggest that the cbZIP domain is a unique DNA-binding domain of the chloroplast protein.

Amino Acid Sequence↗