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At least 145 records · Page 8Linked to original sources
PubMed2000

DNA binding in the central channel of bacteriophage T7 helicase-primase is a multistep process. Nucleotide hydrolysis is not required.

Many helicases assemble into ring-shaped hexamers and bind DNA in their central channel. This raises the question as to how the DNA gets into the central channel to form a topologically linked complex. We have used the presteady-state stopped-flow kinetic method and protein fluorescence changes to investigate the mechanism of single-stranded DNA (ssDNA) binding to the bacteriophage T7 helicase-primase, gp4A'. We have found that the kinetics of 30-mer ssDNA binding to a preformed gp4A' hexamer in the presence of both Mg-dTMP-PCP and Mg-dTTP are similar, indicating that Mg-dTTP binding is sufficient and hydrolysis is not necessary for efficient DNA binding. Multiple transient changes in gp4A' fluorescence revealed a four-step mechanism for DNA binding with Mg-dTTP. These transient changes were analyzed by global fitting and kinetic simulation to determine the intrinsic rate constants of this four-step mechanism. The initial steps, including the bimolecular encounter of the DNA with the helicase and a subsequent conformational change, were fast. We propose that these initial steps of DNA binding occur at a readily accessible site, which is likely to be on the outside of the hexamer ring. The binding of the 30-mer ssDNA at this loading site is followed by slower conformational changes that allow the DNA to transit into the central channel of gp4A' via a ring-opening or threading pathway.

Bacteriophage T7↗
PubMed2002

Transcription coactivator CBP has direct DNA binding activity and stimulates transcription factor DNA binding through small domains.

CBP and p300 are transcriptional coactivators that physically interact with diverse sequence-specific DNA-binding factors through conserved domains. To further investigate the functional roles of these protein-interaction domains in CBP/p300 regulation, we have identified multiple domains of CBP that interact with FKLF2 and the CH2 domain as a new p53 interacting domain of CBP. Functional studies demonstrate that several domains of CBP are capable of stimulating FKLF2 and p53 DNA binding. In addition, we found that CBP through distinct domain is able to bind DNA directly with no specificity. We identified a 51-residue domain in CBP that is capable of interacting with both transcription factors and DNA. We named this domain PDBD for protein and DNA binding domain. These results unveiled two novel activities of CBP. First, these highly conserved domains of CBP not only function to recruit CBP to the target promoter through interaction with DNA-bound transcription factors, but they also actively regulate the DNA binding activity of their interacting factors. Second, by directly interacting with DNA, CBP may orchestrate the formation of stable and promoter-committed transcriptional complexes through interactions with both proteins and promoter DNA.

Amino Acid Sequence↗
PubMed1992

Role of the amino-terminal domain of bacteriophage phi 29 connector in DNA binding and packaging.

The connector of bacteriophage phi 29 is required for prohead assembly, binds DNA, and drives DNA packaging into viral proheads. Limited proteolysis of the connector protein with endoproteinase Glu-C from Staphylococcus aureus V8 and chymotrypsin showed that a domain of the NH2-terminal region is involved in DNA binding and in the subsequent packaging into preformed proheads, but not in prohead assembly. Mutants in specific amino acids of the NH2-terminal domain, obtained by directed mutagenesis techniques, showed that the Ala1-Arg2-Lys3-Arg4 region of the connector is absolutely necessary for DNA packaging into the proheads as well as for efficient DNA binding.

Amino Acid Sequence↗
PubMed2003

DNA-binding and oligomerization studies of the manganese(II) metalloregulatory protein MntR from Bacillus subtilis.

The metalloregulatory protein MntR from Bacillus subtilis acts as a transcriptional regulator of manganese homeostasis. MntR is a member of a subfamily of DtxR-related proteins that perform analogous regulatory functions in a variety of pathogenic organisms. Metal ions activate MntR to bind DNA and repress the transcription of the mntH gene, which encodes for a proton-coupled metal ion transporter. Size-exclusion chromatography and sedimentation equilibrium ultracentrifugation studies show that apo MntR is predominantly a homodimer in solution. Using fluorescence anisotropy measurements, the DNA binding properties of MntR have been examined. In the strict absence of divalent transition metal ions MntR has a low affinity for the mntH control sequence (K(d) > 8.0 microM). However, binding of MntR is stimulated by the presence of Mn(2+) and Cd(2+) to generate high affinity binding with K(d) values of 16.0 and 7.3 nM, respectively. MntR is also shown to bind the mntH control sequence in the presence of other divalent transition metals, including Ni(2+), Cu(2+), and Zn(2+), but with much lower affinity (K(d) approximately 1.3-2.3 microM). The data here demonstrate that differences in metal-activated DNA binding plays a role in the mechanism of manganese(II)-selective transcription factors and that the oligomerization of MntR is metal-independent, which distinguishes this protein from iron(II)-responsive homologues in the DtxR protein family.

Amino Acid Sequence↗
PubMed1980

Identification of a major serum DNA-binding protein as factor B of the alternative complement pathway.

One of the major proteins in human serum capable of binding DNA has been shown to be factor B of the alternative pathway of complement activation. This protein, designated DNA-binding protein-2 (DBP-2), is recognized by antisera directed against both factor B and its activated form, fragment Bb. Its m.w., charge microheterogeneity, and amino acid composition correspond closely with reported values for those properties of factor B. A radioimmunoassay was used to estimate the serum concentration of DBP-2 at 266 +/- 83 microgram/ml, which also corresponds with the level of factor B normally present in the serum. DBP-2 functions as factor B in the activation of factor B-depleted serum. Limited proteolytic treatment of DBP-2 produced a fragment pattern resembling that of factor B both in the m.w. of the fragments and their electrophoretic mobilities. By means of DNA affinity chromatography of the fragments produced by trypsinization, the DNA-binding domain of DBP-2 was localized.

Amino Acid Sequence↗
PubMed1986

DNA-binding domains of human plasma fibronectin. pH and calcium ion modulation of fibronectin binding to DNA and heparin.

We have studied the binding of fibronectin and its thermolysin fragments to DNA and heparin. Elution of polypeptides bound to DNA-cellulose and heparin-Sepharose affinity chromatography columns was performed by NaCl linear gradients in buffers at different pH and in the presence and absence of calcium ions. The NaCl concentration required to elute fibronectin from both types of column increased as the pH decreased. Fibronectin was not retained on DNA-cellulose or heparin-Sepharose affinity chromatography columns using a buffer containing physiological concentrations of Ca2+, Mg2+ and NaCl, at pH 7.4. On the other hand at pH 6.4 in conditions of physiological ionic strength, fibronectin was retained by both columns, eluting from the DNA-cellulose at 280 mM NaCl and from the heparin-Sepharose column at 210 mM. Furthermore, we have studied the interaction of thermolysin-digested fibronectin both with DNA-cellulose and heparin-Sepharose using the above procedure. The results demonstrate that there are four distinct domains, which interact both with DNA and heparin. We also report here the modulation by pH and Ca2+ ions of the interaction with DNA and heparin of these different domains.

Amino Acid Sequence↗
PubMed2001

A novel class of plant-specific zinc-dependent DNA-binding protein that binds to A/T-rich DNA sequences.

Complementary DNA encoding a DNA-binding protein, designated PLATZ1 (plant AT-rich sequence- and zinc-binding protein 1), was isolated from peas. The amino acid sequence of the protein is similar to those of other uncharacterized proteins predicted from the genome sequences of higher plants. However, no paralogous sequences have been found outside the plant kingdom. Multiple alignments among these paralogous proteins show that several cysteine and histidine residues are invariant, suggesting that these proteins are a novel class of zinc-dependent DNA-binding proteins with two distantly located regions, C-x(2)-H-x(11)-C-x(2)-C-x((4-5))-C-x(2)-C-x((3-7))-H-x(2)-H and C-x(2)-C-x((10-11))-C-x(3)-C. In an electrophoretic mobility shift assay, the zinc chelator 1,10-o-phenanthroline inhibited DNA binding, and two distant zinc-binding regions were required for DNA binding. A protein blot with (65)ZnCl(2) showed that both regions are required for zinc-binding activity. The PLATZ1 protein non-specifically binds to A/T-rich sequences, including the upstream region of the pea GTPase pra2 and plastocyanin petE genes. Expression of the PLATZ1 repressed those of the reporter constructs containing the coding sequence of luciferase gene driven by the cauliflower mosaic virus (CaMV) 35S90 promoter fused to the tandem repeat of the A/T-rich sequences. These results indicate that PLATZ1 is a novel class of plant-specific zinc-dependent DNA-binding protein responsible for A/T-rich sequence-mediated transcriptional repression.

AT Rich Sequence↗
PubMed1998

Disruption of target DNA binding in Mu DNA transposition by alteration of position 99 in the Mu B protein.

Target DNA binding by the Mu B protein is an important step in phage Mu transposition; however, the region of Mu B involved in target binding and the mechanism of the interaction are unknown. Previous studies have demonstrated that modification of Mu B with the sulfhydryl-specific reagent N-ethylmaleimide can selectively inhibit target DNA binding. We now show that individual mutation of the three cysteines in Mu B to serine results in proteins which are active in intermolecular strand transfer, but demonstrate variable levels of N-ethylmaleimide resistance. The data indicate that cysteine 99 is the primary site of modification affecting target DNA binding, with a minor contribution resulting from the derivatization of cysteine 129. These findings are confirmed by the construction of Mu B mutants containing a bulky side-chain at the individual cysteine to mimic the N-ethylmaleimide modified protein. The C99Y protein shows a complete loss in target-dependent strand transfer activity under standard reaction conditions and C129Y displays partial activity. The effect of the tyrosine substitutions is specific for target interaction as both mutants show wild-type activity in their ability to stimulate the Mu transposase to perform donor cleavage and intramolecular strand transfer. Finally, a target dissociation assay has shown that the C99Y-DNA complex generated in the presence of ATP-gamma-S has a drastically reduced half-life as previously found for N-ethylmaleimide treated wild-type Mu B. Modification of cysteine 99 is proposed to block target DNA binding by causing steric interference near the DNA binding pocket.

Amino Acid Substitution↗
PubMed1995

Tumor necrosis factor-alpha modulates CCAAT/enhancer binding proteins-DNA binding activities and promotes hepatocyte-specific gene expression during liver regeneration.

Injury-related cytokines, such as tumor necrosis factor-alpha (TNF), may preserve liver-specific gene expression during the subsequent regenerative response by modulating the activity of transcription factors, including CCAAT/enhancer binding proteins (C/EBPs), which regulate differentiated gene expression in hepatocytes. To test this theory, rats were treated with neutralizing antibody to TNF or nonimmune immunoglobulin before partial hepatectomy (PH) and regenerative changes in the messenger RNAs (mRNAs), proteins, and DNA-binding activities of C/EBP isoforms and the expression of a C/EBP-regulated gene, phosphoenol pyruvate carboxykinase (PEPCK), were compared. Before PH, the expressions of C/EBP-alpha, C/EBP-beta, and C/EBP-gamma were similar in the two treatment groups. Dimers containing C/EBP-alpha and C/EBP-beta accounted for virtually all of the C/EBP DNA binding activity and mRNA for PEPCK, the rate limiting hepatocyte enzyme for gluconeogenesis, was barely detected. After PH, in control rats, mRNA and nuclear protein concentrations of C/EBP-beta and C/EBP-gamma increased approximately fivefold by 3 hours after PH. This was accompanied by increased DNA binding activity of these C/EBP isoforms and decreased DNA binding activity of C/EBP-alpha. mRNA levels of PEPCK, a gene that is strongly transactivated by non-alpha C/EBP isoforms, increased fivefold. Pretreatment with anti-TNF antibodies prevented regenerative induction of C/EBP beta and gamma expression and DNA-binding activity. The nature of dimers binding to C/EBP cis-acting elements remained similar to that observed in liver before PH and increases in PEPCK mRNA were blunted. These results support the theory that TNF helps maintain liver-specific gene expression during liver regeneration by altering transcription factor complexes that regulate differentiated gene expression in hepatocytes.

Animals↗
PubMed1982

Immunochemical analysis of the glucocorticoid receptor: identification of a third domain separate from the steroid-binding and DNA-binding domains.

The glucocorticoid-receptor complex can be subdivided into three separate domains by limited proteolysis with trypsin or alpha-chymotrypsin. The following characteristics can be separated: steroid-binding activity (domain A), DNA-binding activity (domain B), and immunoactivity (domain C). We have previously reported the separation of the steroid-binding domain from the DNA-binding domain by limited proteolysis of the receptor with trypsin. In this paper, we report the detection by immunochemical analysis of a third domain of the glucocorticoid receptor, which does not bind hormone. Immunoactivity was detected by using specific antiglucocorticoid receptor antibodies raised in rabbits against purified rat liver glucocorticoid receptor and the assay used was an enzyme-linked immunosorbent assay. After digestion with alpha-chymotrypsin, the immunoactive region of the receptor (domain C) was separated from the other two domains (A and B). The immunoactive fragment was found to have a Stokes radius of 2.6 nm. Further digestion with alpha-chymotrypsin resulted in separation of the immunoactive fragment to give a fragment having a Stokes radius of 1.4 nm. The immunoactive domain could be separated from the half of the glucocorticoid receptor containing the steroid-binding and the DNA-binding domains (Stokes radius, 3.3 nm), by limited proteolysis of the receptor by alpha-chymotrypsin followed by gel filtration or chromatography on DNA-cellulose.

Animals↗
PubMed2005

Xeroderma pigmentosum complementation group E protein (XPE/DDB2): purification of various complexes of XPE and analyses of their damaged DNA binding and putative DNA repair properties.

Xeroderma pigmentosum is characterized by increased sensitivity of the affected individuals to sunlight and light-induced skin cancers and, in some cases, to neurological abnormalities. The disease is caused by a mutation in genes XPA through XPG and the XP variant (XPV) gene. The proteins encoded by the XPA, -B, -C, -D, -F, and -G genes are required for nucleotide excision repair, and the XPV gene encodes DNA polymerase eta, which carries out translesion DNA synthesis. In contrast, the mechanism by which the XPE gene product prevents sunlight-induced cancers is not known. The gene (XPE/DDB2) encodes the small subunit of a heterodimeric DNA binding protein with high affinity to UV-damaged DNA (UV-damaged DNA binding protein [UV-DDB]). The DDB2 protein exists in at least four forms in the cell: monomeric DDB2, DDB1-DDB2 heterodimer (UV-DDB), and as a protein associated with both the Cullin 4A (CUL4A) complex and the COP9 signalosome. To better define the role of DDB2 in the cellular response to DNA damage, we purified all four forms of DDB2 and analyzed their DNA binding properties and their effects on mammalian nucleotide excision repair. We find that DDB2 has an intrinsic damaged DNA binding activity and that under our assay conditions neither DDB2 nor complexes that contain DDB2 (UV-DDB, CUL4A, and COP9) participate in nucleotide excision repair carried out by the six-factor human excision nuclease.

Animals↗
PubMed1993

N-terminal DNA-binding domains contribute to differential DNA-binding specificities of NF-kappa B p50 and p65.

We previously reported that either oxidation or alkylation of NF-kappa B in vitro abrogates DNA binding. We used this phenomenon to help elucidate structural determinants of NF-kappa B binding. We now demonstrate that Cys-62 of NF-kappa B p50 mediates the redox effect and lies within an N-terminal region required for DNA binding but not for dimerization. Several point mutations in this region confer a transdominant negative binding phenotype to p50. The region is highly conserved in all Rel family proteins, and we have determined that it is also critical for DNA binding of NF-kappa B p65. Replacement of the N-terminal region of p65 with the corresponding region from p50 changes its DNA-binding specificity towards that of p50. These data suggest that the N-terminal regions of p50 and p65 are critical for DNA binding and help determine the DNA-binding specificities of p50 and p65. We have defined within the N-terminal region a sequence motif, R(F/G)(R/K)YXCE, which is present in Rel family proteins and also in zinc finger proteins capable of binding to kappa B sites. The potential significance of this finding is discussed.

Amino Acid Sequence↗
PubMed2000

Sequence-specific DNA binding by the glucocorticoid receptor DNA-binding domain is linked to a salt-dependent histidine protonation.

We used isothermal titration calorimetry in the temperature range 21-25 degrees C to investigate the effect of pH on the calorimetric enthalpy (delta H(cal)) for sequence specific DNA-binding of the glucocorticoid receptor DNA-binding domain (GR DBD). Titrations were carried out in solutions containing 100 mM NaCl, 1 mM dithiothreitol, 5% glycerol by volume, and 20 mM Tris, Hepes, Mops, or sodium phosphate buffers at pH 7.5. A strong dependence of delta H(cal) on the buffer ionization enthalpy is observed, demonstrating that the DNA binding of the GR DBD is linked to proton uptake at these conditions. The apparent increase in the pK(a) for an amino acid side chain upon DNA binding is supported by the results of complementary titrations, where delta H(cal) shows a characteristic dependence on the solution pH. delta H(cal) is also a function of the NaCl concentration, with opposite dependencies in Tris and Hepes buffers, respectively, such that a similar delta H(cal) value is approached at 300 mM NaCl. This behavior shows that the DNA-binding induced protonation is inhibited by increased concentrations of NaCl. A comparison with structural data suggests that the protonation involves a histidine (His451) in the GR DBD, because in the complex this residue is located close to a DNA phosphate at an orientation that is consistent with a charged-charged hydrogen bond in the protonated state. NMR spectra show that His451 is not protonated in the unbound protein at pH 7.5. The pH dependence in delta H(cal) can be quantitatively described by a shift of the pK(a) of His451 from approximately 6 in the unbound state to close to 8 when bound to DNA at low salt concentration conditions. A simple model involving a binding competition between a proton and a Na(+) counterion to the GR DBD-DNA complex reproduces the qualitative features of the salt dependence.

Animals↗
PubMed1996

p140/c-Abl that binds DNA is preferentially phosphorylated at tyrosine residues.

EP is a DNA element found in the enhancer and promoter regions of several cellular and viral genes. Previously, we have identified the DNA binding p140/c-Abl protein that specifically recognizes this element. Here we show that phosphorylation is essential for the p140/c-Abl DNA binding activity and for the formation of DNA-protein complexes. Furthermore, by 32P labeling of cells and protein purification, we demonstrate that in vivo the EP-DNA-associated p140/c-Abl is a tyrosine phosphoprotein. By employing two different c-Abl antibodies, we demonstrate the existence of two distinct c-Abl populations in cellular extracts. p140/c-Abl is quantitatively the minor population, is heavily phosphorylated at both serine and tyrosine residues, and is active in autophosphorylation reactions.

Cell Nucleus↗
PubMed1998

Single chain dimers of MASH-1 bind DNA with enhanced affinity.

By designing recombinant genes containing tandem copies of the coding region of the BHLH domain of MASH-1 (MASH-BHLH) with intervening DNA sequences encoding linker sequences of 8 or 17 amino acids, the two subunits of the MASH dimer have been connected to form the single chain dimers MM8 and MM17. Despite the long and flexible linkers which connect the C-terminus of the first BHLH subunit to the N-terminus of the second, a distance of approximately 55 A, the single chain dimers could be produced in Escherichia coli at high levels. MM8 and MM17 were monomeric and no 'cross-folding' of the subunits was observed. CD spectroscopy revealed that, like wild-type MASH-BHLH, MM8 and MM17 adopt only partly folded structures in the absence of DNA, but undergo a folding transition to a mainly alpha-helical conformation on DNA binding. Titrations by electrophoretic mobility shift assays revealed that the affinity of the single chain dimers for E box-containing DNA sequences was increased approximately 10-fold when compared with wild-type MASH-BHLH. On the other hand, the affinity for heterologous DNA sequences was increased only 5-fold. Therefore, the introduction of the peptide linker led to a 4-fold increase in DNA binding specificity from -0.14 to -0.57 kcal/mol.

Amino Acid Sequence↗
PubMed1992

The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted alpha helices: crystal structure of the protein-DNA complex.

The yeast transcriptional activator GCN4 is 1 of over 30 identified eukaryotic proteins containing the basic region leucine zipper (bZIP) DNA-binding motif. We have determined the crystal structure of the GCN4 bZIP element complexed with DNA at 2.9 A resolution. The bZIP dimer is a pair of continuous alpha helices that form a parallel coiled coil over their carboxy-terminal 30 residues and gradually diverge toward their amino termini to pass through the major groove of the DNA-binding site. The coiled-coil dimerization interface is oriented almost perpendicular to the DNA axis, giving the complex the appearance of the letter T. There are no kinks or sharp bends in either bZIP monomer. Numerous contacts to DNA bases and phosphate oxygens are made by basic region residues that are conserved in the bZIP protein family. The details of the bZIP dimer interaction with DNA can explain recognition of the AP-1 site by the GCN4 protein.

Amino Acid Sequence↗
PubMed2003

Identification of novel domains within Sox-2 and Sox-11 involved in autoinhibition of DNA binding and partnership specificity.

Sox transcription factors play key regulatory roles throughout development, binding DNA through a consensus (A/T)(A/T)CAA(A/T)G sequence. Although many different Sox proteins bind to this sequence, it has been observed that gene regulatory elements are commonly responsive to only a small subset of the entire family, implying that regulatory mechanisms exist to permit selective DNA binding and/or transactivation by Sox family members. To identify and explore the mechanisms modulating gene activation by Sox proteins further, we compared the function of Sox-2 and Sox-11. This led to the discovery that Sox proteins are regulated differentially at multiple levels, including transactivation, protein partnerships with Pit-Oct-Unc (POU) transcription factors, and DNA binding autoregulation. Specifically, we determined that Sox-11 activates transcription more strongly than Sox-2 and that the transactivation domain of Sox-11 is primarily responsible for this capability. Additionally, we demonstrate that the Sox-11 DNA binding domain is responsible for selective cooperation with the POU factor Brn-2. This requirement cannot be replaced by the DNA binding domain of Sox-2, indicating that the DNA binding domain of Sox proteins is critical for Sox-POU partnerships. Interestingly, we have also determined that a conserved domain of Sox-11 has the novel capability of autoinhibiting its ability to bind DNA in vitro and to activate gene expression in vivo. Our findings suggest that the autoinhibitory domain can repress promiscuous binding of Sox-11 to DNA and plays an important role in regulating the recruitment of Sox-11 to specific genes.

Binding Sites↗