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PubMedDate not supplied

[Interaction of the antineoplastic agent mitoxantrone with double-stranded nucleic acids].

The binding of mitoxantrone with double-helical nucleic acids was investigated by the methods of isothermal microcalorimetry, circular dichroism and absorption at the ionic strength mu = 0.11 and 0.011 M NaCl at temperature region of 30 divided by 60 degrees C. The investigation shows, that at mu = 0.11 M NaCl mitoxantrone interacts with double-helical nucleic acids in one way only. For such conditions using spectrophotometric titration data Scatchard plots for the binding of mitoxantrone with double-helical nucleic acids were constructed. The calculations show that the saturation stoichiometry is one mitoxantrone molecule per 2 divided by 3 base pairs DNA and 6 divided by 8 base pairs RNA. The dependence of binding constant from GC-content is observed. It is shown that the binding enthalpy of mitoxantrone with DNA and RNA increases linearly and reaches -(3.0 +/- 0.5) kkal per 1 mol mitoxantrone. It is shown that a binding mitoxantrone with double-helical nucleic acids, besides the intercalation of rings, a determinate contribution in the binding is given also by electrostatic interaction of side chains mitoxantrone with nucleic acids.

Animals↗
PubMed2026

African swine fever virus A151R protein antagonizes the antiviral activity of barrier-to-autointegration factor (BAF) by targeting its dsDNA-binding activity.

Barrier-to-autointegration factor (BAF) is a ubiquitous double-stranded DNA-binding protein that compacts DNA and can restrict poxvirus replication in the cytoplasm. BAF antiviral DNA-binding activity is tightly regulated by dynamic phosphorylation mediated by viral and cellular enzymes. For example, vaccinia virus counteracts BAF by encoding the B1 kinase, which phosphorylates BAF and abrogates its DNA-binding activity. Some DNA viruses, such as African swine fever virus (ASFV), undergo cytoplasmic replication but appear to lack a B1-like kinase. Interestingly, ASFV encodes A151R, a viral protein recently found to stably interact with BAF. Here, we demonstrate that A151R is capable of counteracting the antiviral properties of BAF. Structural modeling indicates that A151R is not a protein kinase and does not phosphorylate BAF but instead directly targets its double-stranded DNA-binding interface. This interaction enhances genome replication and progeny production of a B1-deficient virus. Mechanistically, A151R markedly impairs BAF DNA binding and disrupts its dimerization, a key requirement for high-affinity DNA association. Importantly, disruption of the A151R-BAF interaction abolishes these effects and restores BAF antiviral function. In addition, expression of the unphosphorylatable BAF mutant, which normally exhibits strong chromatin association, was redistributed to the cytoplasm in the presence of A151R, further supporting phosphorylation-independent regulation of BAF-DNA association. In conclusion, our findings support a previously unrecognized mechanism by which ASFV A151R disables BAF antiviral activity by obscuring its DNA-binding interface and inhibiting DNA binding in a phosphorylation-independent manner.IMPORTANCEDNA viruses replicating in the cytoplasm must overcome host intrinsic defenses to ensure productive replication, yet the mechanisms underlying their antagonism of the DNA-binding antiviral factor BAF remain incompletely understood. Here, we identify African swine fever virus (ASFV) A151R as a novel viral regulator that disables BAF by targeting its double-stranded DNA-binding interface rather than altering its phosphorylation state. We demonstrate that A151R impairs BAF DNA binding, disrupts its dimerization, and promotes viral DNA accumulation and progeny production in a BAF-dependent manner. Importantly, this activity requires A151R-BAF interaction and is independent of BAF phosphorylation status. Our findings reveal a previously unrecognized strategy employed by ASFV to neutralize host DNA-binding restriction factors and expand the molecular framework of BAF-mediated antiviral defense.

A151R↗
PubMed1997

Analysis of binding interactions in an idiotope-antiidiotope protein-protein complex by double mutant cycles.

The idiotope-antiidiotope complex between the anti-hen egg white lysozyme antibody D1.3 and the anti-D1.3 antibody E5.2 provides a useful model for studying protein-protein interactions. A high-resolution crystal structure of the complex is available [Fields, B. A., Goldbaum, F. A., Ysern, X., Poljak, R.J., & Mariuzza, R. A. (1995) Nature 374, 739-742], and both components are easily produced and manipulated in Escherichia coli. We previously analyzed the relative contributions of individual residues of D1.3 to complex stabilization by site-directed mutagenesis [Dall'Acqua, W., Goldman, E. R., Eisenstein, E., & Mariuzza, R. A. (1996) Biochemistry 35, 9667-9676]. In the current work, we introduced single alanine substitutions in 9 out of 21 positions in the combining site of E5.2 involved in contacts with D1.3 and found that 8 of them play a significant role in ligand binding (delta Gmutant-delta Gwild type > 1.5 kcal/mol). Furthermore, energetically important E5.2 and D1.3 residues tend to be juxtaposed in the crystal structure of the complex. In order to further dissect the energetics of specific interactions in the D1.3-E5.2 interface, double mutant cycles were carried out to measure the coupling of 13 amino acid pairs, 9 of which are in direct contact in the crystal structure. The highest coupling energy (4.3 kcal/mol) was measured for a charged-neutral pair which forms a buried hydrogen bond, while side chains which interact through solvated hydrogen bonds have lower coupling energies (1.3-1.7 kcal/mol), irrespective of whether they involve charged-neutral or neutral-neutral pairs. Interaction energies of similar magnitude (1.3-1.6 kcal/mol) were measured for residues forming only van der Waals contacts. Cycles between distant residues not involved in direct contacts in the crystal structure also showed significant coupling (0.5-1.0 kcal/mol). These weak long-range interactions could be due to rearrangements in solvent or protein structure or to secondary interactions involving other residues.

Antigen-Antibody Complex↗
PubMed2006

Investigation of corannulene for molecular hydrogen storage via computational chemistry and experimentation.

Molecular simulations for hydrogen physisorption with corannulene molecules arranged according to their crystal structure result in good agreement with the weight-percent hydrogen stored as determined experimentally employing a 3-g sample of highly crystalline corannulene at ambient temperatures and 72 bar of pressure. Calculated enthalpies of adsorption for corannulene/hydrogen molecular systems obtained from ab initio calculations which take into account electron correlation via second-order Möller-Plesset perturbation theory are in good agreement with literature experimental enthalpies of adsorption for activated carbons interacting with molecular hydrogen. Ab initio results also show that corannulene molecules arranged in a sandwich structure are important for approximately doubling the binding energy of corannulene interacting with molecular hydrogen through a cooperative interaction. To test the effects of finite temperatures and pressures, stack arrays were used as input for molecular dynamics simulations and indicate that physisorption mechanisms including van der Waals forces and dipole-induced dipole interactions may yield enhanced adsorption capacity in relation to other carbon-based materials. These results will be instrumental in identifying interlayer separations of an array of corannulene or related molecules that may provide a high weight percent of physisorbed hydrogen.

Journal Article↗
PubMed2002

Molecular dynamics simulation of the RNA complex of a double-stranded RNA-binding domain reveals dynamic features of the intermolecular interface and its hydration.

The interaction between double-stranded RNA (dsRNA) and the third double-stranded domain (dsRBD) from Drosophila Staufen protein represents a paradigm to understand how the dsRBD protein family, one of the most common RNA-binding protein units, binds dsRNA. The nuclear magnetic resonance (NMR) structure of this complex and the x-ray structure of another family member revealed the stereochemical basis for recognition, but also raised new questions. Although the crystallographic studies revealed a highly ordered interface containing numerous water-mediated contacts, NMR suggested extensive residual motion at the interface. To address how interfacial motion contributes to molecular recognition in the dsRBD-dsRNA system, we conducted a 2-ns molecular dynamics simulation of the complex derived from Staufen protein and of the separate protein and RNA components. The results support the observation that a high degree of conformational flexibility is retained upon complex formation and that this involves interfacial residues that are critical for dsRBD-dsRNA binding. The structural origin of this residual flexibility is revealed by the analysis of the trajectory of motion. Individual basic side chains switch continuously from one RNA polar group to another with a residence time seldom exceeding 100 ps, while retaining favorable interaction with RNA throughout much of the simulation. Short-lived water molecules mediate some of these interactions for a large fraction of the trajectory studied here. This result indicates that water molecules are not statically associated with the interface, but continuously exchange with the bulk solvent on a 1-10-ps time scale. This work provides new insight into dsRBD-dsRNA recognition and builds upon a growing body of evidence, suggesting that short-lived dynamic interactions play important roles in protein-nucleic acid interactions.

Binding Sites↗
PubMed1983

Interaction of a DNA-binding protein, the product of gene D5 of bacteriophage T5, with double-stranded DNA: effects on T5 DNA polymerase functions in vitro.

The gene D5 product (gpD5) of bacteriophage T5 is a DNA-binding protein that binds preferentially to double-stranded DNA and is essential for T5 DNA replication, yet it inhibits DNA synthesis in vitro. Mechanisms of inhibition were studied by using nicked DNA and primed single-stranded DNA as a primer-template. Inhibition of T5 DNA polymerase activity by gpD5 occurred when double-stranded regions of DNA were saturated with gpD5. The 3' leads to 5' exonuclease associated with T5 DNA polymerase was not very active with nicked DNA, but inhibition of hydrolysis of substituents at 3'-hydroxyl termini by gpD5 could be observed. T5 DNA polymerase appears to be capable of binding to the 3' termini even when double-stranded regions are saturated with gpD5. The interaction of gpD5 with the polymerases at the primer terminus is apparently the primary cause of inhibition of polymerization.

DNA Helicases↗
PubMed1992

Sp1 DNA binding efficiency is highly reduced in nuclear extracts from aged rat tissues.

To explore the role of transcriptional factors in the genesis of the senescent phenotype, nuclear extracts from 4- and 30-month-old rat brains were analyzed for the presence of DNA-binding proteins able to interact with double-stranded oligonucleotides containing recognition sites for sequence-specific DNA-binding factors. Gel shift assays revealed that the DNA-binding efficiency of Sp1 is significantly reduced in aged animals compared to young ones, whereas CTF/NF1 and AP1 from young and old rat nuclear extracts bind their DNA targets with the same efficiency. The quantitative analysis of Sp1 by immunoblotting indicated that equivalent quantities and degrees of heterogeneity of Sp1 protein are present in both nuclear extracts, suggesting that the observed difference is not due to a different expression of this transcriptional factor. DNase I footprinting of the heavy chain ferritin gene promoter, which contains a Sp1 binding site, demonstrated that the nuclear extract from 30-month-old rat brain does not protect the region involved in the regulation of the H ferritin gene by Sp1. This results in a reduction of about 50% of the expression of the H ferritin mRNA in aged rat brains. Furthermore, the Sp1 binding sites present in the SV40 early promoter are not protected in a DNase I footprinting assay where a nuclear extract from 30-month-old rat brain was used as a source of DNA binding proteins. Liver nuclear extracts prepared from young and aged rats demonstrated that a decrease of Sp1 binding efficiency is similarly present in this tissue.

Aging↗
PubMed1998

Direct measurement of the substrate preference of uracil-DNA glycosylase.

Site-directed mutants of the herpes simplex virus type 1 uracil-DNA glycosylase lacking catalytic activity have been used to probe the substrate recognition of this highly conserved and ubiquitous class of DNA-repair enzyme utilizing surface plasmon resonance. The residues aspartic acid-88 and histidine-210, implicated in the catalytic mechanism of the enzyme (Savva, R., McAuley-Hecht, K., Brown, T., and Pearl, L. (1995) Nature 373, 487-493; Slupphaug, G., Mol, C. D., Kavli, B., Arvai, A. S., Krokan, H. E. and Tainer, J. A. (1996) Nature 384, 87-92) were separately mutated to asparagine to allow investigations of substrate recognition in the absence of catalysis. The mutants were shown to be correctly folded and to lack catalytic activity. Binding to single- and double-stranded oligonucleotides, with or without uracil, was monitored by real-time biomolecular interaction analysis using surface plasmon resonance. Both mutants exhibited comparable rates of binding and dissociation on the same uracil-containing substrates. Interaction with single-stranded uracil-DNA was found to be stronger than with double-stranded uracil-DNA, and the binding to Gua:Ura mismatches was significantly stronger than that to Ade:Ura base pairs suggesting that the stability of the base pair determines the efficiency of interaction. Also, there was negligible interaction between the mutants and single- or double-stranded DNA lacking uracil, or with DNA containing abasic sites. These results suggest that it is uracil in the DNA, rather than DNA itself, that is recognized by the uracil-DNA glycosylases.

Catalysis↗
PubMed1980

An equilibrium study of the cooperative binding of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate to the adenosine cyclic 3',5'-monophosphate receptor protein from Escherichia coli.

The binding of adenosine cyclic 3',5'-monophosphate (cAMP) and guanosine cyclic 3',5'-monophosphate (cGMP) to the adenosine cyclic 3',5'-monophosphate receptor protein (CRP) from Escherichia coli was investigated by equilibrium dialysis at pH 8.0 and 20 degrees C at different ionic strengths (0.05--0.60 M). Both cAMP and cGMP bind to CRP with a negative cooperativity that is progressively changed to positive as the ionic strength is increased. The binding data were analyzed with an interactive model for two identical sites and site/site interactions with the interaction free energy--RT ln alpha, and the intrinsic binding constant K and cooperativity parameter alpha were computed. Double-label experiments showed that cGMP is strictly competitive with cAMP, and its binding parameters K and alpha are not very different from that for cAMP. Since two binding sites exist for each of the cyclic nucleotides in dimeric CRP and no change in the quaternary structure of the protein is observed on binding the ligands, it is proposed that the cooperativity originates in ligand/ligand interactions. When bound to double-stranded deoxyribonucleic acid (dsDNA), CRP binds cAMP more efficiently, and the cooperativity is positive even in conditions of low ionic strength where it is negative for the free protein. By contrast, cGMP binding properties remained unperturbed in dsDNA-bound CRP. Neither the intrinsic binding constant K nor the cooperativity parameter alpha was found to be very sensitive to changes of pH between 6.0 and 8.0 at 0.2 M ionic strength and 20 degrees C. For these conditions, the intrinsic free energy and entropy of binding of cAMP are delta H degree = -1.7 kcal . mol-1 and delta S degree = 15.6 eu, respectively.

Animals↗
PubMed2000

The chromosomal protein HMG-D binds to the TAR and RBE RNA of HIV-1.

The high mobility group protein HMG-D is known to bind preferentially to DNA of irregular structures with little or no sequence specificity. Upon binding to DNA, this HMG-box protein widens the minor groove of the double helix and induces a significant bending of the helix. We show here that HMG-D can strongly bind to double-stranded RNA. Electrophoretic mobility shift assays show that HMG-D100 interacts with the transactivation response region (TAR) RNA from HIV-1. Strong interaction with a high affinity Rev protein binding element (RBE) RNA was also characterized. Gel shift experiments performed with several TAR RNA constructs lacking the lateral pyrimidine bulge or with modified apical loop regions indicate that the protein does not recognize the single-strand domains of the RNA but apparently interacts directly with the double-stranded stem regions. No protein-RNA complexes could be detected when using single-stranded oligoribonucleotides. HMG-D protein could bind to the wide minor groove of the A-form TAR RNA. The comparison of the amino acid sequence of HMG-D with that of known RNA binding proteins suggests that the interaction of the protein with a double-stranded RNA implicates the basic region of HMG-D as well as its HMG-box domain. From the in vitro data reported here, we propose a novel functional role for proteins of the HMG-1 family. The results suggest that architectural HMG proteins can be recruited by double-stranded RNA for the development of HIV-1 in the host cell.

Base Sequence↗
PubMed1979

An in vitro study of drug displacement interactions: warfarin-salicylate and warfarin-phenylbutazone.

The binding interactions between warfarin-salicylate and warfarin-phenylbutazone in the presence of 4 g percent bovine serum albumin at 37 degrees C were studied using equilibrium dialysis. Methods of representing and analysing drug binding interactions are discussed. Scatchard plots, double reciprocal plots and the like are shown to be of no use in representing drug displacement interactions since they display only one drug and they can be potentially misleading. It is argued that a preferable method of analysing drug displacement interaction data is in terms of a stepwise multiple equilibria model. The numerical problems associated with fitting this kind of model to the data are discussed. A three-dimensional representation of the binding surface is proposed as a superior means of visualizing drug displacement interactions.

Animals↗
PubMed1997

Characterization of two protein activities that interact at the promoter of the trypanosomatid spliced leader RNA.

All trypanosome mRNAs have a spliced leader (SL). The SL RNA gene in Leptomonas seymouri is a member of the small nuclear RNA gene family. However, the SL RNA is required in stoichiometric amounts for trans-splicing during mRNA formation. Expression of the SL RNA gene requires sequence elements at bp -60 to -70 and bp -30 to -40 upstream from the transcription initiation site. Using conventional and affinity chromatography, we have identified and characterized an approximately 122-kDa protein, promoter-binding protein (PBP) -1, that binds to double-strand DNA. The PBP-1-binding site is within the bp -60 to -70 element determined by DNase I footprinting. Therefore, PBP-1 is the first characterized double-strand DNA binding activity that interacts with a trypanosome gene promoter. A second protein, PBP-2, interacts with the PBP-1:DNA complex and its DNase I footprint extends to include the second promoter element (bp -30 to -40). An alteration of the spacing between the two promoter elements or mutation of the second element decreases PBP-2/PBP-1:DNA stability. Taken together, these data suggest that PBP-1 and PBP-2 are components of a transcription initiation complex that assembles within the SL RNA gene promoter.

Animals↗
PubMed2003

Fabrication of histidine-tagged fusion protein arrays for surface plasmon resonance imaging studies of protein-protein and protein-DNA interactions.

The creation and characterization of histidine-tagged fusion protein arrays using nitrilotriacetic acid (NTA) capture probes on gold thin films for the study of protein-protein and protein-DNA interactions is described. Self-assembled monolayers of 11-mercaptoundecylamine were reacted with the heterobifunctional linker N-succinimidyl S-acetylthiopropionate (SATP) to create reactive sulfhydryl-terminated surfaces. NTA capture agents were immobilized by reacting maleimide-NTA molecules with the sulfhydryl surface. The SATP and NTA attachment chemistry was confirmed with Fourier transform infrared reflection absorption spectroscopy. Oriented protein arrays were fabricated using a two-step process: (i) patterned NTA monolayers were first formed through a single serpentine poly(dimethylsiloxane) microchannel; (ii) a second set of parallel microchannels was then used to immobilize multiple His-tagged proteins onto this pattern at discrete locations. SPR imaging measurements were employed to characterize the immobilization and specificity of His-tagged fusion proteins to the NTA surface. SPR imaging measurements were also used with the His-tagged fusion protein arrays to study multiple antibody-antigen binding interactions and to monitor the sequence-specific interaction of double-stranded DNA with TATA box-binding protein. In addition, His-tagged fusion protein arrays created on gold surfaces were also used to monitor antibody binding with fluorescence microscopy in a sandwich assay format.

Base Sequence↗
PubMed2005

Determinants of specific binding of HMGB1 protein to hemicatenated DNA loops.

Protein HMGB1 has long been known as one of the most abundant non-histone proteins in the nucleus of mammalian cells, and has regained interest recently for its function as an extracellular cytokine. As a DNA-binding protein, HMGB1 facilitates DNA-protein interactions by increasing the flexibility of the double helix, and binds specifically to distorted DNA structures. We have previously observed that HMGB1 binds with extremely high affinity to a novel DNA structure, hemicatenated DNA loops (hcDNA), in which double-stranded DNA fragments containing a tract of poly(CA).poly(TG) form a loop maintained at its base by a hemicatenane. Here, we show that the single HMGB1 domains A and B, the HMG-box domain of sex determination factor SRY, as well as the prokaryotic HMGB1-like protein HU, specifically interact with hcDNA (Kd approximately 0.5 nM). However, the affinity of full-length HMGB1 for hcDNA is three orders of magnitude higher (Kd<0.5 pM) and requires the simultaneous presence of both HMG-box domains A and B plus the acidic C-terminal tail on the molecule. Interestingly, the high affinity of the full-length protein for hcDNA does not decrease in the presence of magnesium. Experiments including a comparison of HMGB1 binding to hcDNA and to minicircles containing the CA/TG sequence, binding studies with HMGB1 mutated at intercalating amino acid residues (involved in recognition of distorted DNA structures), and exonuclease III footprinting, strongly suggest that the hemicatenane, not the DNA loop, is the main determinant of the affinity of HMGB1 for hcDNA. Experiments with supercoiled CA/TG-minicircles did not reveal any involvement of left-handed Z-DNA in HMGB1 binding. Our results point to a tight structural fit between HMGB1 and DNA hemicatenanes under physiological conditions, and suggest that one of the nuclear functions of HMGB1 could be linked to the possible presence of hemicatenanes in the cell.

Binding Sites↗
PubMed1980

The presence of RNA in a double helix inhibits its interaction with histone protein.

The binding of core histones (H2A, H2B, H3, H4) to a circular plasmid DNA and to a circular DNA-RNA hybrid molecule of similar size has been compared. Circular hybrid molecules were formed from single stranded fd DNA by synthesis of the complimentary strand with ribonucleotides using wheat germ RNA polymerase II. Upon reconstitution of plasmid DNA circles with histone, the sedimentation profiles of the DNA remained sharp by increased several fold in rate. Material from the peak fractions of these sedimentations appeared to be condensed circular loops of nucleosomes when examined by electron microscopy (EM), and the mass ratio of DNA to histone (at the histone concentrations which produced the fastest sedimentations) was typical of native chromatin. In contrast, the sedimentation behavior of DNA-RNA hybrid circles after addition of histone remained unchanged except for a minor fraction which exhibited a broad and faster sedimentation rate. Examination by EM revealed that most of the molecules appeared identical to protein free hybrid circles while the minor, faster sedimenting fraction appeared to be two or more circles bound together by protein aggregates. Finally, a linear molecule consisting of about 3000 base pairs of duplex DNA covalently joined on both ends to 1500 base pairs of RNA-DNA hybrid helix was constructed. Reconstitution of this molecule with core histone showed nucleosome formation only on the central DNA duplex region. Isopycnic banding of fixed hybrid-histone mixtures showed that little or no histone had bound to the bulk of the full hybrid molecules. We suggest that the presence of RNA in a nucleic acid duplex inhibits the condensation of the duplex into a nucleosomal structure by histone.

Chromosome Banding↗
PubMed1993

Binding of Ku protein to DNA. Measurement of affinity for ends and demonstration of binding to nicks.

Ku, also known as nuclear Factor IV, is an abundant nuclear DNA-binding protein which requires free DNA ends for the initial interaction with double-stranded DNA (dsDNA) and can bind at multiple sites along dsDNA in an energy-independent manner. Its function in vivo is unknown, but it has been implicated in both DNA replication and repair and in transcriptional control. We have used an electrophoretic mobility shift assay to further define the DNA binding properties of the Ku protein. Titration of Ku to a fixed amount of any of several target linear dsDNA fragments produced ladders of shifted bands proportional to the length of DNA, confirming the multiple binding activity of Ku and demonstrating its sequence-independent nature. Using a short DNA fragment with one Ku binding site, the binding constant of Ku for dsDNA ends was calculated to be 2.4 x 10(9) M-1. Competitive inhibition experiments confirmed the requirement of a free DNA end for binding by Ku and demonstrated that Ku binds isolated nicks in dsDNA. Nick binding was also observed directly using radiolabeled singly nicked circular DNA. The relative affinities of Ku for specific nick sites and free DNA ends were approximately equal, and nick binding was sequence-independent. Finally, in a study of a possible role for Ku in protecting or repairing damaged DNA, Ku was shown to inhibit the ability of T4 DNA ligase to circularize linear dsDNA molecules, demonstrating that some Ku molecules remain at the DNA terminus rather than translocate. A similar inhibition was not observed at nicks. These experiments document a new DNA binding specificity for Ku and further suggest that the high affinity end and nick binding activity is biologically relevant to its functions in vivo.

Animals↗