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PubMed1989

Unspecific DNA binding of the DNA binding domain of the glucocorticoid receptor studied with flow linear dichroism.

The unspecific interaction between the DNA-binding domain of the human glucocorticoid receptor and DNA was studied using linear dichroism (LD) and circular dichroism (CD) spectroscopy. The amplitude of the LD signal was found to increase upon addition of protein at ionic strengths less than 60 nM Na+, indicating an increased persistence length of the complex compared to uncomplexed DNA. Analysis of the LD spectrum suggests that the binding does not involve intercalation of tyrosine residues. Evidence of saturation is found at a binding stoichiometry of approximately 5 DNA base pairs per protein monomer.

DNA↗

PubMed2006

Using evolutionary and structural information to predict DNA-binding sites on DNA-binding proteins.

Proteins that interact with DNA are involved in a number of fundamental biological activities such as DNA replication, transcription, and repair. A reliable identification of DNA-binding sites in DNA-binding proteins is important for functional annotation, site-directed mutagenesis, and modeling protein-DNA interactions. We apply Support Vector Machine (SVM), a supervised pattern recognition method, to predict DNA-binding sites in DNA-binding proteins using the following features: amino acid sequence, profile of evolutionary conservation of sequence positions, and low-resolution structural information. We use a rigorous statistical approach to study the performance of predictors that utilize different combinations of features and how this performance is affected by structural and sequence properties of proteins. Our results indicate that an SVM predictor based on a properly scaled profile of evolutionary conservation in the form of a position specific scoring matrix (PSSM) significantly outperforms a PSSM-based neural network predictor. The highest accuracy is achieved by SVM predictor that combines the profile of evolutionary conservation with low-resolution structural information. Our results also show that knowledge-based predictors of DNA-binding sites perform significantly better on proteins from mainly-alpha structural class and that the performance of these predictors is significantly correlated with certain structural and sequence properties of proteins. These observations suggest that it may be possible to assign a reliability index to the overall accuracy of the prediction of DNA-binding sites in any given protein using its sequence and structural properties. A web-server implementation of the predictors is freely available online at http://lcg.rit.albany.edu/dp-bind/.

Amino Acid Sequence↗

PubMed1985

Conformational change of DNA binding subunit of RNA polymerase II on binding to DNA.

The spatial arrangement of the subunits of RNA polymerase II from Ehrlich ascites tumor cells was investigated by measuring the sensitivity of each subunit in the native enzyme to various proteinases. The results showed that the largest two subunits (a and b) were sensitive to all the proteinases tested, whereas two smaller subunits (e and h) were resistant to these enzymes. These results suggest that in the native enzyme subunits e and h are located in the inside of RNA polymerase II, forming a core. It was also found that the conformation of the DNA binding subunit a changes when the enzyme binds to DNA, and it becomes much more susceptible to chymotryptic digestion.

Animals↗

PubMed2000

The additional 165 amino acids in the B protein of Escherichia coli DNA gyrase have an important role in DNA binding.

DNA gyrase is the only enzyme known to negatively supercoil DNA. The enzyme is a heterotetramer of A(2)B(2) subunit composition. Alignment of the primary sequence of gyrase B (GyrB) from various species shows that they can be grouped into two classes. The GyrB of Gram-negative eubacteria has a stretch of about 165 amino acids in the C-terminal half, which is lacking in other GyrB subunits and type II topoisomerases. In Escherichia coli, no function has so far been attributed to this stretch. In this study, we have tried to assess the function of this region both in vivo and in vitro. A deletant (GyrBDelta160) lacking this region is non-functional in vivo. The holoenzyme reconstituted from gyrase A (GyrA) and GyrBDelta160 shows reduced but detectable supercoiling and quinolone-induced cleavage activity in vitro. GyrBDelta160 retains its ability to bind to GyrA and novobiocin. However, when reconstituted with GyrA, the deletant shows greatly impaired DNA binding. The intrinsic ATPase activity of the GyrBDelta160 is comparable to that of wild type GyrB, but this activity is not stimulated by DNA. These studies indicate that the additional stretch present in GyrB is essential for the DNA binding ability of E. coli gyrase.

Adenosine Triphosphatases↗

PubMed1998

A new model for how O6-methylguanine-DNA methyltransferase binds DNA.

Human methyltransferase (hAT) catalyzes the transfer of an alkyl group from the 6-position of guanine to an active site Cys residue. The physiological role of hAT is the repair of alkylated guanine residues in DNA. However, the repair of methylated or chloroethylated guanine bases negates the effects of certain chemotherapeutic agents. A model of how hAT binds DNA might be useful in the design of compounds that could inactivate hAT. We have used computer modeling studies to generate such a model. The model utilizes a helix-loop-wing DNA binding motif found in Mu transposase. The model incorporates a flipped out guanine base in order to bring the methylated oxygen atom close to the active site Cys residue. The model is consistent with a variety of chemical and biochemical data.

Amino Acid Sequence↗

PubMed1994

DNA-binding-defective mutants of the Epstein-Barr virus lytic switch activator Zta transactivate with altered specificities.

The Epstein-Barr virus BRLF1 and BZLF1 genes are the first viral genes transcribed upon induction of the viral lytic cycle. The protein products of both genes (referred to here as Rta and Zta, respectively) activate expression of other viral genes, thereby initiating the lytic cascade. Among the viral antigens expressed upon induction of the lytic cycle, however, Zta is unique in its ability to disrupt viral latency; expression of the BZLF1 gene is both necessary and sufficient for triggering the viral lytic cascade. We have previously shown that Zta can activate its own promoter (Zp), through binding to two Zta recognition sequences (ZIIIA and ZIIIB). Here we describe mutant Zta proteins that do not bind DNA (referred to as Zta DNA-binding mutants [Zdbm]) but retain the ability to transactivate Zp. Consistent with the inability of these mutants to bind DNA, transactivation of Zp by Zdbm is not dependent on the Zta recognition sequences. Instead, transactivation by Zdbm is dependent upon promoter elements that bind cellular factors. An examination of other viral and cellular promoters identified promoters that are weakly responsive or unresponsive to Zdbm. An analysis of a panel of artificial promoters containing one copy of various promoter elements demonstrated a specificity for Zdbm activation that is distinct from that of Zta. These results suggest that non-DNA-binding forms of some transactivators retain the ability to transactivate specific target promoters without direct binding to DNA.

Amino Acid Sequence↗

PubMed1997

DNA binding and DNA bending by the MelR transcription activator protein from Escherichia coli.

The Escherichia coli melR gene encodes MelR protein which is a member of the AraC/XylS family of bacterial transcription activators. The function of MelR was investigated by making a targeted deletion in the melR gene of the Escherichia coli chromosome. MelR is a transcription activator essential for melibiose- dependent expression of the melAB operon which is needed for bacterial growth with melibiose as a carbon source. To investigate the interactions of MelR at the melAB promoter, both full length MelR and a shortened derivative, MelR173, containing the C-terminal DNA-binding domain, were purified as fusions to glutathione- S -transferase. Circular permutation studies show that both full-length MelR and MelR173 induce an apparent bend upon binding to target sites at the melAB promoter. Bound full-length MelR, but not MelR173, can oligomerise to form larger complexes that are likely to be involved in transcription activation.

Base Sequence↗

PubMed1996

The F plasmid traY gene product binds DNA as a monomer or a dimer: structural and functional implications.

The F factor traY gene product (TraYp) is a site-specific DNA-binding protein involved in initiation of DNA transfer during bacterial conjugation. The sequence of TraYp exhibits a unique direct-repeat structure predicted to have a ribbon-helix-helix DNA-binding motif in each repeat unit. The stoichiometry of TraYp binding to DNA was determined to further support the hypothesis that TraYp is a member of the ribbon-helix-helix family of DNA-binding proteins. A glutathione-S-transferase-traY fusion protein was purified and shown to possess almost wild-type DNA-binding activity. DNA-binding experiments were performed in which the DNA ligand was incubated with either the fusion protein, the wild-type protein, or both. The results indicate that TraYp can bind DNA as a monomer or a dimer. Thus a TraYp monomer folds into a stable three-dimensional structure similar to that of a dimer of the ribbon-helix-helix proteins Arc or Mnt. A homology model of a TraYp monomer has been constructed using the co-crystal structure of Arc bound to DNA as a template to provide additional support for this conclusion. In addition, we have shown that an origin of the transfer-deletion mutant lacking approximately half of the TraYp-binding site can only be bound by a monomer of TraYp. The functional implications of this result are discussed.

Amino Acid Sequence↗

PubMed2003

Using electrostatic potentials to predict DNA-binding sites on DNA-binding proteins.

A method to detect DNA-binding sites on the surface of a protein structure is important for functional annotation. This work describes the analysis of residue patches on the surface of DNA-binding proteins and the development of a method of predicting DNA-binding sites using a single feature of these surface patches. Surface patches and the DNA-binding sites were initially analysed for accessibility, electrostatic potential, residue propensity, hydrophobicity and residue conservation. From this, it was observed that the DNA-binding sites were, in general, amongst the top 10% of patches with the largest positive electrostatic scores. This knowledge led to the development of a prediction method in which patches of surface residues were selected such that they excluded residues with negative electrostatic scores. This method was used to make predictions for a data set of 56 non-homologous DNA-binding proteins. Correct predictions made for 68% of the data set.

Binding Sites↗

PubMed2001

Thermodynamics of specific and nonspecific DNA binding by two DNA-binding domains conjugated to fluorescent probes.

The complexes designed in this work combine the sequence-specific binding properties of helix-turn-helix DNA-binding motifs with intercalating cyanine dyes. Thermodynamics of the Hin recombinase and Tc3 transposase DNA-binding domains with and without the conjugated dyes were studied by fluorescence techniques to determine the contributions to specific and nonspecific binding in terms of the polyelectrolyte and hydrophobic effects. The roles of the electrostatic interactions in binding to the cognate and noncognate sequences indicate that nonspecific binding is more sensitive to changes in salt concentration, whereas the change in the heat capacity shows a greater sensitivity to temperature for the sequence-specific complexes in each case. The conjugated dyes affect the Hin DNA-binding domain by acting to anchor a short stretch of amino acids at the N-terminal end into the minor groove. In contrast, the N-terminal end of the Tc3 DNA-binding domain is bound in a well-ordered fashion to the DNA even in the absence of the conjugated dye. The conjugated dye and the DNA-binding domain portions of each conjugate bind noncooperatively to the DNA. The characteristic thermodynamic parameters of specific and nonspecific DNA binding by each of the DNA-binding domains and their respective conjugates are presented.

Amino Acid Sequence↗

PubMed2003

Adenovirus type 5 DNA binding protein stimulates binding of DNA polymerase to the replication origin.

The adenovirus (Ad) DNA-binding protein (DBP) is essential for the elongation phase of Ad DNA replication by unwinding the template in an ATP-independent fashion, employing its capacity to form multimers. DBP also enhances the rate of initiation, with the highest levels obtained at low concentrations of Ad DNA polymerase (Pol). Here, we show that stimulation of initiation depends on the template conformation. Maximal stimulation, up to 15-fold, is observed on double-stranded or viral TP-containing origins. The stimulation is reduced on partially single-stranded origins and DBP does not enhance initiation any more once the origin is completely unwound. This suggests a role for DBP in origin unwinding that is comparable to its unwinding capacity during elongation. However, mutant DBP proteins defective in unwinding and elongation can still enhance initiation on ds templates. DBP also stimulates the binding of nuclear factor I (NFI) to the origin and lowers the K(m) for coupling of the first nucleotide to the precursor terminal protein by Pol. Mobility shift experiments reveal that DBP stimulates the binding of Pol on double-stranded origin and nonorigin DNA but not on single-stranded DNA. This effect is specific for DBP and is also seen with other DNA Pols. Our results suggest that, rather than by origin unwinding, DBP enhances initiation by modulating the origin conformation such that DNA Pol can bind more efficiently.

Adenoviridae↗

PubMed1995

Reduction of chromium(VI) by ascorbate leads to chromium-DNA binding and DNA strand breaks in vitro.

Chromium(VI) is a known human carcinogen which requires intracellular reduction for activation. Ascorbate (vitamin C) has been reported to function as a major reductant of Cr(VI) in animals and cell culture systems. The reaction of Cr(VI) with varying concentrations of ascorbate was studied under physiological conditions in vitro in order to determine the types of reactive intermediates produced and to evaluate the reactivity of these intermediates with DNA. Reactions of 1.8 mM Cr(VI) with 0-18 mM ascorbate at pH 7.0 in N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES; 0.10 M) and tris(hydroxymethyl)aminomethane hydrochloride (Tris.HCl; 0.050 M) buffers were studied by electron paramagnetic resonance and UV/visible spectroscopy. Cr(V) and carbon-based free radical adducts of 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) were observed at 0.5 to 1 and 1 to 1 reactions of ascorbate to Cr(VI). Levels of Cr(V) were higher for reactions in HEPES buffer, and levels of carbon-based radicals were higher in Tris.HCl buffer. Levels of Cr(IV) and Cr(III) increased with increasing concentration of ascorbate in both buffers. Reaction of Cr(VI) with varying ascorbate in the presence of calf thymus DNA or pBR322 DNA resulted in Cr-DNA adducts and plasmid relaxation, respectively. Maximum binding of Cr to DNA was observed for the 1:1 reaction ratio of Cr(VI) with ascorbate in both HEPES and Tris.HCl buffers, but total Cr bound to DNA was 8-fold lower in Tris.HCl than HEPES buffer. Preincubation of Cr(VI) with ascorbate before reaction with DNA decreased Cr-DNA binding to background levels. Preincubation of Cr(III) with ascorbate resulted in only low Cr-DNA binding. Levels of Cr-DNA binding were higher with single-stranded vs double-stranded DNA. Reactions with 14C-labeled ascorbate produced no cross-linking of ascorbate to DNA. Maximum plasmid relaxation was observed for the 1:1 ascorbate to Cr(VI) ratio in both buffers; however, single-strand breaks were 2-fold higher in Tris.HCl than HEPES buffer. Reactions with plasmid in the presence of DMPO quenched formation of single-strand breaks. Interpretation of these results in light of the spectroscopic studies suggested that Cr(V) and carbon-based radicals were responsible for Cr-DNA adducts and DNA single-strand breaks, respectively.

Ascorbic Acid↗

PubMed2004

Residues required for Bacillus subtilis PhoP DNA binding or RNA polymerase interaction: alanine scanning of PhoP effector domain transactivation loop and alpha helix 3.

Bacillus subtilis PhoP is a member of the OmpR family of response regulators that activates or represses genes of the Pho regulon upon phosphorylation by PhoR in response to phosphate deficiency. Because PhoP binds DNA and is a dimer in solution independent of its phosphorylation state, phosphorylation of PhoP may optimize DNA binding or the interaction with RNA polymerase. We describe alanine scanning mutagenesis of the PhoP alpha loop and alpha helix 3 region of PhoPC (Val190 to E214) and functional analysis of the mutated proteins. Eight residues important for DNA binding were clustered between Val202 and Arg210. Using in vivo and in vitro functional analyses, we identified three classes of mutated proteins. Class I proteins (PhoP(I206A), PhoP(R210A), PhoP(L209A), and PhoP(H208A)) were phosphorylation proficient and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Class II proteins (PhoP(H205A) and PhoP(V204A)) were phosphorylation proficient and could dimerize but could not bind DNA prior to phosphorylation. Members of this class had higher transcription activation in vitro than in vivo. The class III mutants, PhoP(V202A) and PhoP(D203A), had a reduced rate of phosphotransfer and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Seven alanine substitutions in PhoP (PhoP(V190A), PhoP(W191A), PhoP(Y193A), PhoP(F195A), PhoP(G197A,) PhoP(T199A), and PhoP(R200A)) that specifically affected transcription activation were broadly distributed throughout the transactivation loop extending from Val190 to as far toward the C terminus as Arg200. PhoP(W191A) and PhoP(R200A) could not activate transcription, while the other five mutant proteins showed decreased transcription activation in vivo or in vitro or both. The mutagenesis studies may indicate that PhoP has a long transactivation loop and a short alpha helix 3, more similar to OmpR than to PhoB of Escherichia coli.

Alanine↗

PubMed1999

PBX and MEIS as non-DNA-binding partners in trimeric complexes with HOX proteins.

HOX, PBX, and MEIS transcription factors bind DNA through a homeodomain. PBX proteins bind DNA cooperatively as heterodimers with MEIS family members and also with HOX proteins from paralog groups 1 to 10. MEIS proteins cooperatively bind DNA with ABD-B class HOX proteins of groups 9 and 10. Here, we examine aspects of dimeric and higher-order interactions between these three homeodomain classes. The most significant results can be summarized as follows. (i) Most of PBX N terminal to the homeodomain is required for efficient cooperative binding with HOXD4 and HOXD9. (ii) MEIS and PBX proteins form higher-order complexes on a heterodimeric binding site. (iii) Although MEIS does not cooperatively bind DNA with ANTP class HOX proteins, it does form a trimer as a non-DNA-binding partner with DNA-bound PBX-HOXD4. (iv) The N terminus of HOXD4 negatively regulates trimer formation. (v) MEIS forms a similar trimer with DNA-bound PBX-HOXD9. (vi) A related trimer (where MEIS is a non-DNA-binding partner) is formed on a transcriptional promoter within the cell. (vii) We observe an additional trimer class involving non-DNA-bound PBX and DNA-bound MEIS-HOXD9 or MEIS-HOXD10 heterodimers that is enhanced by mutation of the PBX homeodomain. (viii) In this latter trimer, PBX is likely to contact both MEIS and HOXD9/D10. (ix) The stability of DNA binding by all trimers is enhanced relative to the heterodimers. These findings suggest novel functions for PBX and MEIS in modulating the function of DNA-bound MEIS-HOX and PBX-HOX heterodimers, respectively.

Antennapedia Homeodomain Protein↗

PubMed1992

DNA-binding and non-DNA-binding forms of the transformed glucocorticoid receptor.

In this work we have probed the mechanism responsible for two non-DNA-binding states of the mouse glucocorticoid receptor. In the first case, transformed receptors were treated with hydrogen peroxide. It is known that oxidizing agents promote the formation of disulfide bonds in the glucocorticoid receptor, but it has not been determined what domains are involved in any disulfide bond formation that leads to inactivation of DNA-binding activity. We show here that hydrogen peroxide inhibits DNA-binding by the 15-kDa tryptic fragment containing the DNA-binding fingers with the same concentration dependency as it inhibits DNA-binding by the uncleaved receptor. This suggests that all of the effect of peroxide is on sulfhydryl groups within the zinc fingers. After dissociation (transformation) of cytosolic heteromeric glucocorticoid receptor complexes, only a portion (40-60%) of the dissociated receptors can bind to DNA-cellulose. We show that the 15-kDa tryptic fragment derived from the portion of transformed receptors that do not bind to DNA is itself competent at DNA-binding.

Animals↗

PubMed2000

Functional mapping of the MH1 DNA-binding domain of DPC4/SMAD4.

The transcription factor Smad4 binds DNA in response to a TGF-beta ligand-initiated intracellular signaling cascade. SMAD4 is deleted or mutated during tumorigenesis in many human tumors. Some of these mutations occur in the N-terminal portion of the protein, the Mad homology 1 (MH1) region, which exhibits sequence-specific DNA-binding. We used alanine scanning mutagenesis and natural mutations to map the subregion of the MH1 domain necessary for that function. We created 20 individual mutations in the MH1 region of human Smad4 and assayed their effect on DNA-binding in vitro. Mutation of residues in the less conserved N- and C-terminal areas of the MH1 region had no effect on DNA-binding. However, mutations in the domain from L43 to R135 caused a dramatic reduction of the ability of Smad4 to bind DNA. Previous work demonstrated a beta-hairpin protein motif within this region to be responsible for DNA-binding, but suggested that the tumorigenic mutations occurring outside this motif may target a separate function of the MH1 domain. Our results demonstrate that the MH1 domain as a whole is very sensitive to changes in overall structure, and that tumorigenic mutations within the region of L43-R135 indeed would target DNA-binding.

Amino Acid Sequence↗

PubMed1999

Reduction of G-box binding factor DNA binding activity, but not G-box binding factor abundance, causes the downregulation of RBCS2 expression during early tomato fruit development.

The downregulation of RBCS2 promoter activity during tomato fruit development has been investigated by transient gene expression. A major drop in promoter activity occurs between 5 and 25 mm fruit diameter, corresponding to the late cell division to early cell enlargement phase. This drop is abolished by a mutation of the single G-box element necessary for high RBCS2 promoter activity in young tomato fruit. The G-box binding activity of fruit nuclear and total protein extracts drops concomitantly with the reduction of RBCS2 promoter activity while G-box binding factor expression is not affected. The data indicate that the developmental signal that downregulates the RBCS2 promoter acts on the regulation of DNA binding activity of constitutively expressed G-box binding factors.

Arabidopsis↗

PubMed2003

The role of intercalating residues in chromosomal high-mobility-group protein DNA binding, bending and specificity.

Ubiquitous high-mobility-group (HMGB) chromosomal proteins bind DNA in a non-sequence- specific fashion to promote chromatin function and gene regulation. Minor groove DNA binding of the HMG domain induces substantial DNA bending toward the major groove, and several interfacial residues contribute by DNA intercalation. The role of the intercalating residues in DNA binding, bending and specificity was systematically examined for a series of mutant Drosophila HMGB (HMG-D) proteins. The primary intercalating residue of HMG-D, Met13, is required both for high-affinity DNA binding and normal DNA bending. Leu9 and Tyr12 directly interact with Met13 and are required for HMG domain stability in addition to linear DNA binding and bending, which is an important function for these residues. In contrast, DNA binding and bending is retained in truncations of intercalating residues Val32 and Thr33 to alanine, but DNA bending is decreased for the glycine substitutions. Furthermore, substitution of the intercalating residues with those predicted to be involved in the specificity of the HMG domain transcription factors results in increased DNA affinity and decreased DNA bending without increased specificity. These studies reveal the importance of residues that buttress intercalating residues and suggest that features of the HMG domain other than a few base-specific hydrogen bonds distinguish the sequence-specific and non-sequence-specific HMG domain functions.

Amino Acid Sequence↗