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PubMed2000

Structural homology between DNA binding sites of DNA polymerase beta and DNA topoisomerase II.

Unsaturated long-chain fatty acids selectively bind to the DNA binding sites of DNA polymerase beta and DNA topoisomerase II, and inhibit their activities, although the amino acid sequences of these enzymes are markedly different from each other. Computer modeling analysis revealed that the fatty acid interaction interface in both enzymes has a group of four amino acid residues in common, forming a pocket which binds to the fatty acid molecule. The four amino acid residues were Thr596, His735, Leu741 and Lys983 for yeast DNA topoisomerase II, corresponding to Thr79, His51, Leu11 and Lys35 for rat DNA polymerase beta. Using three-dimensional structure model analysis, we determined the spatial positioning of specific amino acid residues binding to the fatty acids in DNA topoisomerase II, and subsequently obtained supplementary information to build the structural model.

Amino Acid Sequence↗

PubMed1999

Signal-dependent DNA binding and functional domains of the quorum-sensing activator TraR as identified by repressor activity.

TraR, a member of the LuxR family of quorum-sensing transcription factors, is responsible for the population density-dependent regulation of Ti plasmid conjugal transfer. The protein requires as coinducer an acyl-homoserine lactone signal molecule called AAI (Agrobacterium autoinducer) that is produced by the bacteria themselves. TraR only activates its target genes, making it difficult to determine whether interaction with AAI is required for binding DNA or for initiating transcription. To assess this, we converted TraR into a repressor by placing a copy of the tra box, an 18-bp inverted repeat believed to be the recognition site for this protein, over the -10 region of a promoter driving expression of lacZ. Repression of this promoter by TraR depended on AAI or, at higher concentrations, VAI, the closely related signal of Vibrio fischeri. C-terminal deletions as short as 2 aa and N-terminal deletions as short as 4 aa in TraR abolished both repressor and activator functions. The C-terminal mutants were strongly dominant over TraR, suggesting that they can form heteromultimers with the wild-type activator. Mutants of TraR with substitutions at Asp-10 and Gly-123 failed to activate a positively controlled reporter but continued to repress the chimeric promoter in an AAI-dependent manner. We conclude that TraR recognizes the tra box as its binding site, that binding of TraR to this site depends on AAI, and that the N-terminal half of the protein contains one or more domains that are required for activation but not for multimerization, for interaction with the acyl-homoserine lactone, or for DNA binding.

Agrobacterium tumefaciens↗

PubMed1998

Cooperative DNA-binding by Bicoid provides a mechanism for threshold-dependent gene activation in the Drosophila embryo.

The Bicoid morphogen directs pattern formation along the anterior-posterior (A-P) axis of the Drosophila embryo. Bicoid is distributed in a concentration gradient that decreases exponentially from the anterior pole, however, it transcribes target genes such as hunchback in a step-function-like pattern; the expression domain is uniform and has a sharply defined posterior boundary. A 'gradient-affinity' model proposed to explain Bicoid action states that (i) cooperative gene activation by Bicoid generates the sharp on/off switch for target gene transcription and (ii) target genes with different affinities for Bicoid are expressed at different positions along the A-P axis. Using an in vivo yeast assay and in vitro methods, we show that Bicoid binds DNA with pairwise cooperativity; Bicoid bound to a strong site helps Bicoid bind to a weak site. These results support the first aspect of the model, providing a mechanism by which Bicoid generates sharp boundaries of gene expression. However, contrary to the second aspect of the model, we find no significant difference between the affinity of Bicoid for the anterior gene hunchback and the posterior gene knirps. We propose, instead, that the arrangement of Bicoids bound to the target gene presents a unique signature to the transcription machinery that, in combination with overall affinity, regulates the extent of gene transcription along the A-P axis.

Animals↗

PubMed1998

A disorder-to-order transition coupled to DNA binding in the essential zinc-finger DNA-binding domain of yeast ADR1.

The motional dynamics and solvent-exchange behavior of free and DNA-bound forms of the minimal zinc-finger DNA-binding domain of the yeast transcription factor ADR1 (ADR1-DBD) are investigated using NMR. The parameters measured include the 1H-15N heteronuclear NOE, 15N and 1H T1 relaxation rates, 15N T2 relaxation rates, and solvent-exchange rates. The spin relaxation parameters, spectral density maps, and solvent-exchange behavior show that, exclusive of the N and C termini, three distinct regions of free ADR1-DBD exhibit different motions on multiple timescales. The N-terminal proximal, or accessory, region appears to be unstructured and highly flexible: it exhibits large amplitude motions on a picosecond timescale, little or no protection from solvent exchange, and random-coil proton chemical shifts. The two zinc fingers tumble anisotropically as folded domains, with the tumbling of the individual fingers being only partly correlated to each other, and are modestly protected from solvent exchange except near the tips of the fingers and in the linker joining them. Free ADR1-DBD exhibits exchange broadening around P97 in the proximal region, at the tip of finger 1, and throughout finger 2. Upon binding, most of the proximal region and both zinc fingers tumble as a single domain and exhibit significantly reduced picosecond timescale motions. This region becomes more protected from solvent exchange. The bound portion of the proximal region is proposed to lie exposed on the surface of the DNA. Exchange broadening remains around P97 but also becomes evident for residues in direct contact with the DNA and in the linker. We conclude that the region of ADR1-DBD essential for high-affinity binding undergoes a disorder-to-order transition upon binding to its cognate DNA and, together with the zinc fingers, forms a cohesive molecular complex with the nucleic acid.

Binding Sites↗

PubMed1997

Two distinct factor-binding DNA elements in cardiac myosin light chain 2 gene are essential for repression of its expression in skeletal muscle. Isolation of a cDNA clone for repressor protein Nished.

The expression of the cardiac myosin light chain 2 (MLC2) gene is repressed in skeletal muscle as a result of the negative regulation of its transcription. Two regulatory elements, the cardiac specific sequence (CSS) located upstream (-360 base pairs) and a downstream negative modulatory sequence (NMS), which function in concert with each other, are required for repression of the MLC2 promoter activity in skeletal muscle. Individually, CSS and NMS have no effect. Transient transfection analysis with recombinant plasmids indicated that CSS- and NMS-mediated repression of transcription is position- and orientation-dependent and is transferable to heterologous promoters. A minimal conserved motif, GAAG/CTTC, present in both CSS and NMS, is responsible for repression as the mutation in the core CTTC sequence alone was sufficient to abrogate its repressor activity. The DNA binding assay by gel mobility shift analysis revealed that one of the two complexes, CSSBP2, is significantly enriched in embryonic skeletal muscle relative to cardiac muscle. In extracts from adult skeletal muscle, where the cardiac MLC2 expression is suppressed, both complexes, CSSBP1 and CSSBP2, were present, whereas the cardiac muscle extracts contained CSSBP1 alone, suggesting that the protein(s) in the CSSBP2 complex accounts for the negative regulation of cardiac MLC2 in skeletal muscle. A partial cDNA clone (Nished) specific for the candidate repressor factor was isolated by expression screening of the skeletal muscle cDNA library by multimerized CSS-DNA as probe. The recombinant Nished protein binds to the CSS-DNA, but not to DeltaCSS-DNA where the core CTTC sequence was mutated. The amino acid sequence of Nished showed a significant structural similarity to the sequence of transcription factor "runt," a known repressor of gap and pair-rule gene expression in Drosophila.

Amino Acid Sequence↗

PubMed1985

Site-specific DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein.

The bacteriophage SP01 genome encodes a virus-specific type II DNA-binding protein, TF1. The bacterial proteins of this ubiquitous and evolutionarily conserved class are thought to bind non-specifically to DNA. In contrast, the experiments described here demonstrate that TF1 binds to specific sites in SP01 DNA. Several of these sites have been characterized by DNase I 'footprinting' and four of them have been shown to overlap strong phage promoters for Bacillus subtilis RNA polymerase holoenzyme. We speculate on the possible structural basis of site-selective DNA binding by a protein of this class.

Bacteriophages↗

PubMed1993

Interaction of the C-terminal region of p105 with the nuclear localisation signal of p50 is required for inhibition of NF-kappa B DNA binding activity.

DNA binding of the homodimeric p50 subunit of NF-kappa B was inhibited by a bacterially expressed protein containing the ankyrin repeats present in the C-terminus of the p105 precursor but not by the I kappa B protein MAD-3. However p50 was retained on protein affinity matrices containing either the C-terminal ankyrin repeats of p105 or MAD-3. To investigate the interaction between p50 and proteins containing ankyrin repeats we have used a number of approaches to probe the accessibility of the p50 nuclear localisation signal in the protein complex. A monoclonal antibody recognising a linear epitope either very close to, or including, the nuclear localisation signal of the p50 protein could immunoprecipitate p50 homodimers but was unable to precipitate the protein when it was bound to the C-terminal region of p105. A close association between the nuclear localisation signal of p50 and the C-terminal region of p105 was also suggested by protease accessibility experiments. While the nuclear localisation signal of free p50 is extremely susceptible to cleavage with trypsin the same site is masked in the presence of the C-terminal ankyrin repeats of p105 and, to a lesser extent MAD-3. Removal of the nuclear localisation signal by trypsin digestion generates a protein that is fully competent for DNA binding but is refractile to inhibition by the C-terminal ankyrin repeats of p105. Addition of DNA destabilises complexes between p50 and ankyrin repeat containing proteins, increasing the susceptibility of the nuclear localisation signal to trypsin cleavage. The data suggest that there is a rapid exchange of p50 between complexes containing DNA or I kappa B proteins via a metastable complex containing DNA, p50 and I kappa B.

Amino Acid Sequence↗

PubMed1999

Mechanism of DNA binding by the DnaB helicase of Escherichia coli: analysis of the roles of domain gamma in DNA binding.

We have analyzed the mechanism of single-stranded DNA (ssDNA) binding mediated by the C-terminal domain gamma of the DnaB helicase of Escherichia coli. Sequence analysis of this domain indicated a specific basic region, "RSRARR", and a leucine zipper motif that are likely involved in ssDNA binding. We have carried out deletion as well as in vitro mutagenesis of specific amino acid residues in this region in order to determine their function(s) in DNA binding. The functions of the RSRARR domain in DNA binding were analyzed by site-directed mutagenesis. DnaBMut1, with mutations R(328)A and R(329)A, had a significant decrease in the DNA dependence of ATPase activity and lost its DNA helicase activity completely, indicating the important roles of these residues in DNA binding and helicase activities. DnaBMut2, with mutations R(324)A and R(326)A, had significantly attenuated DNA binding as well as DNA-dependent ATPase and DNA helicase activities, indicating that these residues also play a role in DNA binding and helicase activities. The role(s) of the leucine zipper dimerization motif was (were) determined by deletion analysis. The DnaB Delta 1 mutant with a 55 amino acid C-terminal deletion, which left the leucine zipper and basic DNA binding regions intact, retained DNA binding as well as DNA helicase activities. However, the DnaB Delta 2 mutant with a 113 amino acid C-terminal deletion that included the leucine zipper dimerization motif, but not the RSRARR sequence, lost DNA binding, DNA helicase activities, and hexamer formation. The major findings of this study are (i) the leucine zipper dimerization domain, I(361)-L(389), is absolutely required for (a) dimerization and (b) ssDNA binding; (ii) the base-rich RSRARR sequence is required for DNA binding; (iii) three regions of domain gamma (gamma I, gamma II, and gamma III) differentially regulate the ATPase activity; (iv) there are likely three ssDNA binding sites per hexamer; and (v) a working model of DNA unwinding by the DnaB hexamer is proposed.

Adenosine Triphosphatases↗

PubMed1993

Reversal of resistance to adriamycin by 8-chloro-cyclic AMP in adriamycin-resistant HL-60 leukemia cells is associated with reduction of type I cyclic AMP-dependent protein kinase and cyclic AMP response element-binding protein DNA-binding activities.

8-Chloro-cyclic AMP (8-Cl-cAMP) produces growth-inhibitory and differentiating activity in the promyelocytic leukemia cell line HL-60. Adriamycin (ADR)-resistant HL-60 (HL-60/AR) cells exhibit the multidrug-resistant phenotype but do not express the mdr1 gene product P-glycoprotein. To explore potential signaling processes that may be involved in this atypical form of drug resistance, 8-Cl-cAMP was used as a modulator of the cAMP second messenger signal transduction pathway. Treatment for 48 hr with a 10% inhibitory concentration of 8-Cl-cAMP potentiated ADR cytotoxicity 14-fold in HL-60/AR cells but not in the parental cell line. 8-Cl-cAMP was stable to hydrolysis in the medium after 48 hr and was present intracellularly predominantly as phosphorylated metabolites (70%) and the parent compound (30%). No difference occurred in ADR accumulation in HL-60/AR cells after treatment with 8-Cl-cAMP. Accompanying the 8-Cl-cAMP-mediated increase in ADR cytotoxicity in HL-60/AR cells was a reduction in the cytosolic type I cAMP-dependent protein kinase (PKA) and disappearance of the nuclear PKA holoenzyme. Coincident with these changes in drug-resistant cells was a marked reduction in the DNA-binding activity of the cAMP response element-binding protein to levels equivalent to those in sensitive cells. This effect appears to result from reduced phosphorylation of the cAMP response element-binding protein. These results suggest that the potentiation by 8-Cl-cAMP of ADR cytotoxicity in HL-60/AR cells occurs through down-regulation of nuclear type I PKA and cAMP response element-binding factors whose activities are regulated by PKA.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1992

DNA binding to mouse cells is mediated by cell-surface molecules: the role of these DNA-binding molecules as target antigens in murine lupus.

Autoimmunity to a 28-29-kDa cell-surface DNA-binding molecule has previously been described in patients with systemic lupus erythematosus and related autoimmune diseases. This report describes experiments that implicate a similar antigen-antibody system in the evolution of autoimmunity in lupus-prone mice. DNA binding to murine spleen cells was found to be a saturable phenomenon that was inhibited by excess cold DNA and trypsinization. The role of autoimmunity to murine cell-surface DNA-binding molecules in lupus-prone mice (MRL lpr/lpr, MRL +/+, BXSB) was compared to normal mice (BALB/c, C3H.SW) by means of an assay that measured the inhibition of cell-surface DNA binding. Only sera from lupus strains had inhibitory activity and this component was shown to be an IgM autoantibody. Furthermore, we isolated a spontaneously occurring IgM monoclonal antibody from the spleen of an MRL/lpr mouse, which inhibited DNA binding to mouse cells. Time-course studies indicated that young female MRL/lpr mice lacked detectable activity against cell-surface DNA-binding molecules; however, by 8-10 weeks maximal inhibitory activity was observed. This response occurred prior to the development of significant antinuclear antibody activity. With the appearance of overt disease and anti-DNA antibodies, inhibition of DNA-binding activity became undetectable. These findings mirror previous studies on autoimmunity to a cell-surface DNA-binding molecule on human leucocytes, but have the added advantage of permitting the study of the temporal evolution of this inhibitory activity in relation to disease expression.

Animals↗

PubMed1993

Identification of AML-1 and the (8;21) translocation protein (AML-1/ETO) as sequence-specific DNA-binding proteins: the runt homology domain is required for DNA binding and protein-protein interactions.

The AML1 gene on chromosome 21 is disrupted in the (8;21)(q22;q22) translocation associated with acute myelogenous leukemia and encodes a protein with a central 118-amino-acid domain with 69% homology to the Drosophila pair-rule gene, runt. We demonstrate that AML-1 is a DNA-binding protein which specifically interacts with a sequence belonging to the group of enhancer core motifs, TGT/cGGT. Electrophoretic mobility shift analysis of cell extracts identified two AML-1-containing protein-DNA complexes whose electrophoretic mobilities were slower than those of complexes formed with AML-1 produced in vitro. Mixing of in vitro-produced AML-1 with cell extracts prior to gel mobility shift analysis resulted in the formation of higher-order complexes. Deletion mutagenesis of AML-1 revealed that the runt homology domain mediates both sequence-specific DNA binding and protein-protein interactions. The hybrid product, AML-1/ETO, which results from the (8;21) translocation and retains the runt homology domain, both recognizes the AML-1 consensus sequence and interacts with other cellular proteins.

Animals↗

PubMed2000

Crystal structure of the HIV-1 integrase catalytic core and C-terminal domains: a model for viral DNA binding.

Insolubility of full-length HIV-1 integrase (IN) limited previous structure analyses to individual domains. By introducing five point mutations, we engineered a more soluble IN that allowed us to generate multidomain HIV-1 IN crystals. The first multidomain HIV-1 IN structure is reported. It incorporates the catalytic core and C-terminal domains (residues 52-288). The structure resolved to 2.8 A is a Y-shaped dimer. Within the dimer, the catalytic core domains form the only dimer interface, and the C-terminal domains are located 55 A apart. A 26-aa alpha-helix, alpha6, links the C-terminal domain to the catalytic core. A kink in one of the two alpha6 helices occurs near a known proteolytic site, suggesting that it may act as a flexible elbow to reorient the domains during the integration process. Two proteins that bind DNA in a sequence-independent manner are structurally homologous to the HIV-1 IN C-terminal domain, suggesting a similar protein-DNA interaction in which the IN C-terminal domain may serve to bind, bend, and orient viral DNA during integration. A strip of positively charged amino acids contributed by both monomers emerges from each active site of the dimer, suggesting a minimally dimeric platform for binding each viral DNA end. The crystal structure of the isolated catalytic core domain (residues 52-210), independently determined at 1.6-A resolution, is identical to the core domain within the two-domain 52-288 structure.

Amino Acid Sequence↗

PubMed1997

Sequences flanking the core DNA-binding domain of bovine papillomavirus type 1 E2 contribute to DNA-binding function.

We have compared a series of molecular constructs that contain the minimal DNA-binding and dimerization domain of bovine papillomavirus type 1 (BPV-1) E2 alone or this binding domain plus the adjacent 16 or 40 amino acids to test the role of the flanking sequences in E2 function. The presence of these sequences resulted in an up to eightfold increase in the affinity of E2 for its target DNA and stabilized the protein against denaturation both in the absence of DNA and in the form of DNA-protein complexes. In addition, an aspartic acid-to-tyrosine mutation within the flanking region blocked DNA binding and function. These data demonstrate that sequences flanking the core domain contribute to E2 function and are, in fact, an integral part of the DNA-binding domain of BPV-1 E2.

Animals↗

PubMed1996

DNA-binding determinants of the alpha subunit of RNA polymerase: novel DNA-binding domain architecture.

The Escherichia coli RNA polymerase alpha-subunit binds through its carboxy-terminal domain (alpha CTD) to a recognition element, the upstream (UP) element, in certain promoters. We used genetic and biochemical techniques to identify the residues in alpha CTD important for UP-element-dependent transcription and DNA binding. These residues occur in two regions of alpha CTD, close to but distinct from, residues important for interactions with certain transcription activators. We used NMR spectroscopy to determine the secondary structure of alpha CTD, alpha CTD contains a nonstandard helix followed by four alpha-helices. The two regions of alpha CTD important for DNA binding correspond to the first alpha-helix and the loop between the third and fourth alpha-helices. The alpha CTD DNA-binding domain architecture is unlike any DNA-binding architecture identified to date, and we propose that alpha CTD has a novel mode of interaction with DNA. Our results suggest models for alpha CTD-DNA and alpha CTD-DNA-activator interactions during transcription initiation.

Amino Acid Sequence↗

PubMed1992

Conserved cysteine residue in the DNA-binding domain of the bovine papillomavirus type 1 E2 protein confers redox regulation of the DNA-binding activity in vitro.

The bovine papillomavirus type 1 E2 open reading frame encodes three proteins involved in viral DNA replication and transcriptional regulation. These polypeptides share a carboxyl-terminal domain with a specific DNA-binding activity; through this domain the E2 polypeptides form dimers. In this study, we demonstrate the inhibition of E2 DNA binding in vitro by reagents that oxidize or otherwise chemically modify the free sulfhydryl groups of reactive cysteine residues. However, these reagents had no effect on DNA-binding activity when the E2 polypeptide was first bound to DNA, suggesting that the free sulfhydryl group(s) may be protected by DNA binding. Sensitivity to sulfhydryl modification was mapped to a cysteine residue at position 340 in the E2 DNA-binding domain, an amino acid that is highly conserved among the E2 proteins of different papillomaviruses. Replacement of this residue with other amino acids abrogated the sensitivity to oxidation-reduction changes but did not affect the DNA-binding property of the E2 protein. These results suggest that papillomavirus DNA replication and transcriptional regulation could be modulated through the E2 proteins by changes in the intracellular redox environment. Furthermore, a motif consisting of a reactive cysteine residue carboxyl-terminal to a lysine residue in a basic region of the DNA-binding domain is a feature common to a number of transcriptional regulatory proteins that, like E2, are subject to redox regulation. Thus, posttranslational regulation of the activity of these proteins by the intracellular redox environment may be a general phenomenon.

Amino Acid Sequence↗

PubMed1983

Mutations in lambda repressor's amino-terminal domain: implications for protein stability and DNA binding.

The DNA binding properties of 52 different single-amino acid substitutions in lambda repressor's amino-terminal domain have been characterized. Seven proteins bearing mutations that change solvent-exposed side chains have been purified. The amino-terminal domains of these mutant repressors are folded and are comparable to the wild-type amino-terminal domain in thermal stability. In contrast, a purified mutant repressor bearing a substitution in a buried side chain contains an amino-terminal domain with decreased thermal stability. We argue that mutations that alter solvent-exposed wild-type side chains define residues that form the operator DNA binding surface of lambda repressor whereas completely or partially buried mutations exert their effect by decreasing protein stability.

Amino Acid Sequence↗

PubMed1989

Two-dimensional 1H nuclear magnetic resonance studies on the gene V-encoded single-stranded DNA-binding protein of the filamentous bacteriophage IKe. I. Structure elucidation of the DNA-binding wing.

Two-dimensional nuclear magnetic resonance techniques were used to obtain residue- and sequence-specific assignments in the 1H spectrum of the single-stranded DNA-binding protein encoded by gene V of the filamentous phage IKe (IKe GVP). The residue-specific assignments are based on the analysis of J-correlated spectra, i.e. correlated spectroscopy and homonuclear-Hartmann-Hahn total correlated spectroscopy. Complete assignments of side-chain spin systems, e.g. long side-chains, were, to a major part, derived from two-dimensional spectra obtained by means of the latter technique. Sequence-specific residue assignments were obtained for the two neighbouring residues V41 and Y42, and the amino acid sequence segment encompassing residues S17 through I29. The structure of this segment, a beta-loop, was deduced from the interresidue nuclear Overhauser effect pattern. Residues S17 through V19 and P26 through I29 form an anti-parallel beta-ladder segment, whereas residues Q21 to K25 constitute the loop region. The beta-loop is expected to project into the solution and is intimately involved in binding to single-stranded DNA; it is therefore designated the "DNA-binding wing". By analogy with the structure of the DNA-binding wing deduced from IKe GVP, a similar structure is proposed for the corresponding domain of the gene V protein encoded by the filamentous phage Ff for which, from X-ray diffraction studies, a three-dimensional structure has been deduced. Essential differences appear to exist between the DNA-binding domain in the X-ray structure and that proposed in this paper. Possible reasons for these differences are discussed.

Amino Acid Sequence↗

PubMed2004

From RPA to BRCA2: lessons from single-stranded DNA binding by the OB-fold.

Recent years have witnessed tremendous progress in our structural and biophysical understanding of how replication protein A (RPA), a major nuclear ssDNA-binding protein (SSB), binds DNA. The four ssDNA-binding domains of RPA have the characteristic OB (oligonucleotide/oligosaccharide-binding) fold and contact DNA with specific polarity via a hierarchy-driven dynamic pathway. A growing mass of data suggest that many aspects of the ssDNA binding mechanism are conserved among SSBs of different origin. However, this conservation is not restricted to the SSB class. The concepts of ssDNA binding by the OB-fold, first derived from the RPA structure, have been successfully applied to the functional characterization of the BRCA2 (breast cancer susceptibility gene 2) protein. The BRCA2 structure, in its turn, has helped to better understand RPA function.

BRCA2 Protein↗