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At least 37 records · Page 2Linked to original sources
PubMed1992

Identification of a 60-kilodalton Rb-binding protein, RBP60, that allows the Rb-E2F complex to bind DNA.

Several reports have indicated that the product of the retinoblastoma gene (Rb) complexes with the transcription factor E2F. We present evidence that the DNA-binding of the Rb-E2F complex involves another cellular factor. Addition of Rb to purified preparations of E2F does not generate an Rb-E2F complex that can bind DNA, and in fact, we see an inhibition of the DNA-binding ability of E2F. On the other hand, addition of Rb to cruder preparations of E2F results in the formation of an Rb-E2F complex (E2Fr) that can bind DNA and produces a distinct complex in gel retardation assays. We have identified and purified a 60-kDa protein that allows the Rb-E2F complex to bind DNA, and we show that this 60-kDa protein exerts its effect by directly interacting with Rb.

Animals↗
PubMed1998

The DNA binding domain of the human c-Abl tyrosine kinase preferentially binds to DNA sequences containing an AAC motif and to distorted DNA structures.

The c-Abl tyrosine kinase protein is implicated in the signaling pathway as well as in transcription, DNA repair, apoptosis, and several other vital biological processes essential for cell proliferation or differentiation. The interaction of c-Abl with DNA is important for some of these functions, but the exact nature of this interaction is still a matter of controversy. The present study addresses the DNA-binding properties of the human c-Abl protein. Using CASTing experiments, the consensus binding site 5'-AA/CAACAAA/C was determined. The central highly conserved AAC triplet appears to constitute the crucial core element in the binding sequences of the c-Abl protein. The c-Abl DNA-binding domain recognizes specific sequences and interacts with deformed DNA structures such as four-way junctions and bubble DNA containing a large single-stranded loop, as determined by electromobility shift assay, melting temperature studies, and binding to specific oligonucleotides covalently linked to beads. Additional competition experiments suggest that the interaction mainly involves contacts within the minor groove of the double helix. The DNA-binding properties of c-Abl are reminiscent of those of high-mobility group (HMG)-like proteins such as LEF-1 and SRY. However, the circular permutation and ring closure assays and DNA unwinding experiments reveal that, unlike HMGs, c-Abl does not bend its target sequence. In addition, it is shown that the protein potentiates the DNA relaxation activity of topoisomerase I. These findings indicate that the interaction of c-Abl with DNA is both sequence-selective and structure-dependent.

Base Composition↗
PubMed1990

Folding transition in the DNA-binding domain of GCN4 on specific binding to DNA.

Protein-DNA recognition is often mediated by a small domain containing a recognizable structural motif, such as the helix-turn-helix or the zinc-finger. These motifs are compact structures that dock against the DNA double helix. Another DNA recognition motif, found in a highly conserved family of eukaryotic transcription factors including C/EPB, Fos, Jun and CREB, consists of a coiled-coil dimerization element the leucine-zipper and an adjoining basic region which mediates DNA binding. Here we describe circular dichroism and 1H-NMR spectroscopic studies of another family member, the yeast transcriptional activator GCN4. The 58-residue DNA-binding domain of GCN4, GCN4-p, exhibits a concentration-dependent alpha-helical transition, in accord with previous studies of the dimerization properties of an isolated leucine-zipper peptide. The GCN4-p dimer is approximately 70% helical at 25 degrees C, implying that the basic region adjacent to the leucine zipper is largely unstructured in the absence of DNA. Strikingly, addition of DNA containing a GCN4 binding site (AP-1 site) increases the alpha-helix content of GNC4-p to at least 95%. Thus, the basic region acquires substantial alpha-helical structure when it binds to DNA. A similar folding transition is observed on GCN4-p binding to the related ATF/CREB site, which contains an additional central base pair. The accommodation of DNA target sites of different lengths clearly requires some flexibility in the GCN4 binding domain, despite its high alpha-helix content. Our results indicate that the GCN4 basic region is significantly unfolded at 25 degrees C and that its folded, alpha-helical conformation is stabilized by binding to DNA.

Activating Transcription Factors↗
PubMed1995

Criteria for the mode of binding of DNA binding agents.

A complete characterization of DNA binding agents requires that their mode of binding to DNA be established. In the absence of high resolution structural data, the mode of binding is, of necessity, usually inferred indirectly from various solution studies. The purpose of this study is to show that only certain methods can be used reliably to infer the DNA binding mode. Comparative fluorescence and hydrodynamic studies using the proven intercalator ethidium and the groove binder Hoechst 33258 are described. The results of our studies show that while fluorescence intensity, polarization, and quenching measurements can detect a binding interaction of the ligand with DNA, none are sensitive indicators of the binding mode. Fluorescence contact energy transfer studies can reliably indicate intercalation, as can viscosity measurements. Our results illustrate reliable criteria that may be used to distinguish intercalation from groove binding in the absence of high resolution structural data.

Animals↗
PubMed1993

Conformational shifts propagate from the oligomerization domain of p53 to its tetrameric DNA binding domain and restore DNA binding to select p53 mutants.

p53 is a conformationally flexible sequence-specific DNA binding protein mutated in many human tumors. To understand why the mutant p53 proteins associated with human tumors fail to bind DNA, we mapped the DNA binding domain of wild-type p53 and examined its regulation by changes in the protein conformation. Using site-directed mutagenesis, residues 90-286 of mouse p53 were shown to form the sequence-specific DNA binding domain. Two highly conserved regions within this domain, regions IV and V, were implicated in contacting DNA. Wild-type p53 bound DNA as a tetramer, each subunit recognizing five nucleotides of the 20 nucleotide-long DNA site. Conformational shifts of the oligomerization domain propagated to the tetrameric DNA binding domain, regulating DNA binding activity, but did not affect the subunit stoichiometry of wild-type p53 oligomers. Interestingly, conformational shifts could also be propagated within certain p53 mutants, rescuing DNA binding. One of these mutants was the mouse equivalent of human histidine 273, which is frequently associated with human tumors.

Amino Acid Sequence↗
PubMed1999

Effects of nucleoside analog incorporation on DNA binding to the DNA binding domain of the GATA-1 erythroid transcription factor.

We investigate here the effects of the incorporation of the nucleoside analogs araC (1-beta-D-arabinofuranosylcytosine) and ganciclovir (9-[(1,3-dihydroxy-2-propoxy)methyl] guanine) into the DNA binding recognition sequence for the GATA-1 erythroid transcription factor. A 10-fold decrease in binding affinity was observed for the ganciclovir-substituted DNA complex in comparison to an unmodified DNA of the same sequence composition. AraC substitution did not result in any changes in binding affinity. 1H-15N HSQC and NOESY NMR experiments revealed a number of chemical shift changes in both DNA and protein in the ganciclovir-modified DNA-protein complex when compared to the unmodified DNA-protein complex. These changes in chemical shift and binding affinity suggest a change in the binding mode of the complex when ganciclovir is incorporated into the GATA DNA binding site.

Animals↗
PubMed1997

acrB mutation located at carboxyl-terminal region of gyrase B subunit reduces DNA binding of DNA gyrase.

Mutations that exhibit susceptibility to acriflavine have been isolated and classified as acr mutations in Escherichia coli. We cloned the acrB gene, which has been identified as a mutation of the gyrB gene, and found a double point mutation altering two consecutive amino acids (S759R/R760C) in the COOH-terminal region of the gyrase B subunit. The mutant B subunit was found to associate with the A subunit to make the quaternary structure, and the reconstituted gyrase showed an 80-fold reduction of specific activity in DNA supercoiling assay; the sensitivity to acriflavine was not different in the same unit of wild-type and mutant gyrases. The mutant enzyme retained intrinsic ATPase activity, but DNA-dependent stimulation was observed infrequently. A gel shift assay showed that acriflavine inhibited the DNA binding of gyrase. The acrB mutation also reduced significantly the DNA binding of gyrase but did not change the sensitivity to acriflavine. These results revealed that the acrB mutation is related to the inhibitory mechanism of acriflavine; and the acriflavine sensitivity of the mutant, at least in vitro, is caused mainly by reduction of the enzyme activity. Further, our findings suggest that the COOH-terminal region of the B subunit is essential for the initial binding of gyrase to the substrate DNA.

Amino Acid Sequence↗
PubMed1993

Ligand effects on platinum binding to DNA. A comparison of DNA binding properties for cis- and trans-[PtCl2(amine)2] (amine = NH3, pyridine).

The DNA binding properties of cis- and trans-[PtCl2(pyridine)2] have been examined and compared with their NH3 analogs, cis- and trans-DDP. The presence of a planar ligand reduces the rates of DNA binding but does not greatly affect the overall conformation of CT DNA, as measured by circular dichroism spectroscopy. The sequence specificity of trans-[PtCl2(py)2] includes alternating purine-pyrimidine sequences. The sequence specificity is further different between the two pyridine isomers, and the steric effects of two cis-pyridine groups are demonstrated by the appearance of relatively few binding sites in the 49-bp duplex. The effects of the pyridine ligand are further manifested by a greatly enhanced DNA-DNA interstrand cross-linking efficiency for the trans isomer, with a cross-link per adduct frequency of between 0.14 and 0.23, depending on the rb of the sample. The unwinding of closed circular pUC19 DNA by trans-[PtCl2(pyridine)2] is also more efficient than that by either DDP isomer, with an unwinding angle calculated at phi = 17 degrees (compare cis-DDP with phi = 13 degrees and trans-DDP with phi = 9-10 degrees). In contrast, little unwinding is induced by cis-[PtCl2(pyridine)2], with phi = 4 degrees. These results in particular invert the standard cis/trans structure-activity relationships observed previously for [PtCl2(NH3)2]. The results are discussed with respect to the previously demonstrated effect of activation of the trans-platinum geometry using sterically hindered ligands.

Base Sequence↗
PubMed1988

Effects of in vitro dephosphorylation on DNA-binding and DNA helicase activities of simian virus 40 large tumor antigen.

Simian virus 40 large T antigen is a phosphoprotein with two clusters of phosphorylation sites. Each cluster includes four serine residues and one threonine residue. In vitro treatment with intestinal alkaline phosphatase removes the phosphate groups from the serine but not from the threonine residues. Potato acid phosphatase additionally dephosphorylates the phosphothreonine (Thr-124) in the N-terminal cluster but does not attack the phosphothreonine in the C-terminal cluster (Thr-701). Two biochemical functions of untreated and partially dephosphorylated T antigen were assayed, namely, its specific DNA-binding property and its DNA helicase activity. After treatment with alkaline phosphatase, T antigen had a severalfold higher affinity for the specific binding sites in the viral genomic control region, in particular, for binding site II in the origin of replication. However, T antigen, when dephosphorylated by acid phosphatase, had DNA-binding properties similar to those of the untreated control. Neither alkaline nor acid dephosphorylation affected the DNA helicase activity of T antigen.

Acid Phosphatase↗
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↗
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↗
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↗
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↗
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↗