B-DNA Binding Research Articles

BRCA2 C-terminal clamp restructures RAD51 dimers to bind B-DNA for replication fork stability.

Tumor suppressor protein BRCA2 acts with RAD51 in replication-fork protection (FP) and homology-directed DNA break repair (HDR). Critical for cancer etiology and therapy resistance, BRCA2 C-terminus was thought to stabilize RAD51-filaments after they assemble on single-stranded (ss)DNA. Here we determined the detailed crystal structure for BRCA2 C-terminal interaction-domain (TR2i) with ATP-bound RAD51 prior to DNA binding. In contrast to recombinogenic RAD51-filaments comprising extended ATP-bound RAD51 dimers, TR2i unexpectedly reshapes ATP-RAD51 into a unique dimer conformation accommodating double-stranded B-DNA binding unsuited for HDR initiation. Structural, biochemical, and molecular results with interface-guided mutations uncover TR2i's FP mechanism. Proline-driven secondary-structure stabilizes residue triads and spans the RAD51 dimer engaging pivotal interactions of RAD51 M210 and BRCA2 S3291/P3292, the cyclin-dependent kinase (CDK) phosphorylation site that toggles between FP during S-phase and HDR in G2. TR2i evidently acts as an allosteric clamp switching RAD51 from ssDNA to double-stranded and B-DNA binding enforcing FP over HDR.

Dual conformational recognition by Z-DNA binding protein is important for the B-Z transition process.

Left-handed Z-DNA is radically different from the most common right-handed B-DNA and can be stabilized by interactions with the Zα domain, which is found in a group of proteins, such as human ADAR1 and viral E3L proteins. It is well-known that most Zα domains bind to Z-DNA in a conformation-specific manner and induce rapid B–Z transition in physiological conditions. Although many structural and biochemical studies have identified the detailed interactions between the Zα domain and Z-DNA, little is known about the molecular basis of the B–Z transition process. In this study, we successfully converted the B–Z transition-defective Zα domain, vvZαE3L, into a B–Z converter by improving B-DNA binding ability, suggesting that B-DNA binding is involved in the B–Z transition. In addition, we engineered the canonical B-DNA binding protein GH5 into a Zα-like protein having both Z-DNA binding and B–Z transition activities by introducing Z-DNA interacting residues. Crystal structures of these mutants of vvZαE3L and GH5 complexed with Z-DNA confirmed the significance of conserved Z-DNA binding interactions. Altogether, our results provide molecular insight into how Zα domains obtain unusual conformational specificity and induce the B–Z transition.

Binding Behaviors for Different Types of DNA G‐Quadruplexes: Enantiomers of [Ru(bpy)2(L)]2+ (L=dppz, dppz‐idzo)

Polymorphic DNA G-quadruplex recognition has attracted great interest in recent years. The strong binding affinity and potential enantioselectivity of chiral [Ru(bpy)2 (L)](2+) (L=dipyrido[3,2-a:2',3'-c]phenazine, dppz-10,11-imidazolone; bpy=2,2'-bipyridine) prompted this investigation as to whether the two enantiomers, Δ and Λ, can show different effects on diverse structures with a range of parallel, antiparallel and mixed parallel/antiparallel G-quadruplexes. These studies provide a striking example of chiral-selective recognition of DNA G-quadruplexes. As for antiparallel (tel-Na(+)) basket G-quadruplex, the Λ enantiomers bind stronger than the Δ enantiomers. Moreover, the behavior reported here for both enantiomers stands in sharp contrast to B-DNA binding. The chiral selectivity toward mixed parallel/antiparallel (tel-K(+)) G-quadruplex of both compounds is weak. Different loop arrangements can change chiral complex selectivity for both antiparallel and mixed parallel/antiparallel G-quadruplex. Whereas both Δ and Λ isomers bind to parallel G-quadruplexes with comparable affinity, no appreciable stereoselective G-quadruplex binding of the isomers was observed. In addition, different binding stoichiometries and binding modes for Δ and Λ enantiomers were confirmed. The results presented here indicate that chiral selective G-quadruplex binding is not only related to G-quadruplex topology, but also to the sequence and the loop constitution.

Klotho Prevents NFκB Translocation and Protects Endothelial Cell From Senescence Induced by Uremia.

In patients with renal disease, uremia raises oxidative stress and senescence in endothelial cells, which can lead to endothelial dysfunction and cardiovascular disease. Klotho protein is a β-glucuronidase capable of hydrolyzing steroid β-glucuronides. This protein is recognized as an antiaging gene, that modulate both stress-induced senescence and functional response. The aim of the study was to investigate how senescence and oxidative stress induced by uremia in endothelial cells affects Klotho expression and whether intra or extracellular Klotho has effects on the response of these cells. Senescence and oxidative stress was obtained by exposure to uremic serum. Telomere length, the enzyme β-galactosidase, and oxidative stress were studied by flow cytometry. Nuclear factor kappa B activity was determined by electrophoretic mobility shift assay. The expression of Klotho decreased with the uremia and preceded the manifestations of cell aging. Levels of intracellular Klotho decreases associated to endothelial senescence, and exogenous Klotho prevents cellular senescence by inhibiting the increase in oxidative stress induced by uremia and diminished the nuclear factor kappa B-DNA binding ability.

Solution structure of the Zbeta domain of human DNA-dependent activator of IFN-regulatory factors and its binding modes to B- and Z-DNAs.

The DNA-dependent activator of IFN-regulatory factors (DAI), also known as DLM-1/ZBP1, initiates an innate immune response by binding to foreign DNAs in the cytosol. For full activation of the immune response, three DNA binding domains at the N terminus are required: two Z-DNA binding domains (ZBDs), Zα and Zβ, and an adjacent putative B-DNA binding domain. The crystal structure of the Zβ domain of human DAI (hZβ(DAI)) in complex with Z-DNA revealed structural features distinct from other known Z-DNA binding proteins, and it was classified as a group II ZBD. To gain structural insights into the DNA binding mechanism of hZβ(DAI), the solution structure of the free hZβ(DAI) was solved, and its bindings to B- and Z-DNAs were analyzed by NMR spectroscopy. Compared to the Z-DNA-bound structure, the conformation of free hZβ(DAI) has notable alterations in the α3 recognition helix, the "wing," and Y145, which are critical in Z-DNA recognition. Unlike some other Zα domains, hZβ(DAI) appears to have conformational flexibility, and structural adaptation is required for Z-DNA binding. Chemical-shift perturbation experiments revealed that hZβ(DAI) also binds weakly to B-DNA via a different binding mode. The C-terminal domain of DAI is reported to undergo a conformational change on B-DNA binding; thus, it is possible that these changes are correlated. During the innate immune response, hZβ(DAI) is likely to play an active role in binding to DNAs in both B and Z conformations in the recognition of foreign DNAs.

Abstract 3674: Inhibition of noncanonical NF-kappa B activation by (−)-DHMEQ in vitro and in adult T-cell leukemia cells

Abstract [Objective and Rationale]Noncanonical NF-kappa B consisting of RelB and p52 is especially important for the maturation of B-lymphocytes and autoimmune diseases. In one hand, we have designed and synthesized a novel NF-kappa B inhibitor (−)-DHMEQ. It showed potent anti-inflammatory and anti cancer effects in animal models without any toxicity. We have reported (−)-DHMEQ covalently binds to the specific Cys residue of each NF-kappa B component and that it inhibits the DNA binding of canonical NF-kappa B components. However, the effects on noncanonical NF-kappa B has not been studied at the molecular level. In the present research we have studied theinhibitory effect of (−)-DHMEQ on noncanonical NF-kappa B in vitro and in cultured lymphoma cells in which noncanonical NF-kappa B is constitutively activated. [Experimental Design and Results]We prepared recombinant noncanonical NF-kappa B proteins, RelB and p52. (−)-DHMEQ was shown to covalently bind to Cys144 of RelB but not to p52 in MLADI-TOF/MS and mutation analysis. Either RelB or p52 does not form homodimer. Then, we prepared HeLa cells over–expressing full length RelB and p52. (−)-DHMEQ inhibited the NF-kappa B activity in RelB(wild type)/p52 cells but not in RelB(C144S)/p52 expressing cells in EMSA. Thus (−)-DHMEQ was shown to target Cys 144 of RelB in cultured cells to inhibit noncanonical NF-kappa B-DNA binding. It also inhibited RelB/p52 binding to DNA in vitro. Then, we looked for the cell line in which noncanonical NF-kappa B is constitutively activated using anti-RelB in EMSA. MT-1 cells are HTLV-1 transformed T-cells and often used for the adult T-cell leukemia (ATL) model. We found that the highly activated NF-kappa B is mainly consisting with noncanonical one in MT-1 cells. (−)-DHMEQ effectively inhibited the noncanonical NF-kappa B activity in MT-1 cells. [Conclusion] (−)-DHMEQ binds to RelB and effectively inhibited RelB/p52-DNA binding. We have found noncanonical NF-kappa B is highly activated ATL-like MT-1 cells, which was inhibited by (−)-DHMEQ. (−)-DHMEQ may be useful for the treatment of ATL and other diseases induced by noncanonical NF-kappaB activation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3674.

Theoretical models of pentamidine analogs activity based on their DNA minor groove complexes

The B-DNA binding property of pentamidine analogs was analyzed employing molecular dynamic simulations and UV–vis absorption spectroscopy. Their AATT and ATAT sequence preferences were also examined. In theoretical energy computations, we considered two solvation models: the implicit model I, where the solvent is characterized by a dielectric constant and the explicit model E, where water molecules are treated as discrete entities. The binding energy correlates well with the increase of melting temperature of the 5′-d(CGCGAATTCGCG) 2-3′ and 5′-d(CGCGATATCGCG) 2-3′ dodecamers after complexation with ligands (Δ T m ), and deduced models predicted adequately the DNA affinities of the studied diamidines.

Recognition of B-DNA by Neomycin−Hoechst 33258 Conjugates

Recent developments have indicated that aminoglycoside binding is limited not to RNA but to nucleic acids that, like RNA, adopt conformations similar to the A-form. We have further sought to expand the utility of aminoglycoside binding to B-DNA structures by conjugating neomycin, an aminoglycoside antibiotic, with the B-DNA minor groove binding ligand Hoechst 33258. Described herein are novel neomycin-Hoechst 33258 conjugates developed for exploring B-DNA groove recognition. We have varied the two reported conjugates in linker length and composition in an effort to improve our understanding of the spatial differences that define B-DNA binding. Spectroscopic studies such as ultraviolet (UV) melting, isothermal fluorescence titrations, differential scanning calorimetry (DSC), and circular dichroism (CD) together illustrate the mode of binding by such conjugates. Both conjugates exhibit enhanced thermal stabilization of A.T rich duplexes when compared to Hoechst 33258.

Metal ions alter lipopolysaccharide‐induced NFκB binding in monocytes

Metals are components of a variety of biomaterials used in orthopedic and dental appliances; however, their biocompatibility with the surrounding tissues is not completely understood. Monocytes are important immune cells that respond to inflammatory stimuli by rapidly producing a variety of inflammatory proteins. Regulation of this response often involves activation of the transcription factor NF kappa B. The current study was designed to determine whether monocyte activation of NF kappa B in response to bacterial lipopolysaccharide (LPS) is affected by pretreatment with metal ions. Concentrations of metal ions that affected cell number after 24 h of exposure were first determined. Then THP-1 human monocytes were cultured for 2 h in media containing metal ions at concentrations below levels that altered cell growth. Parallel cultures were treated with 10 microg/mL Escherichia coli LPS, and all samples were cultured an additional 2 h. Nuclear proteins were extracted and normalized amounts were incubated with [(32)P]-end-labeled NF kappa B consensus oligonucleotide. NF kappa B-DNA complexes were identified and quantified by electrophoretic mobility shift analysis. The extent of NF kappa B-DNA complex formation after metal ion pretreatment with or without LPS induction was compared to no treatment or LPS-only treated controls. Finally, LPS-induced IL1 beta secretion was measured from palladium-treated and control cells. Concentrations were identified for each metal ion (Ag(+), Co(2+), Cu(2+), Hg(2+), Ni(2+), and Pd(2+)) that did not reduce cell number after 24 h of exposure (ranging from 5 microM for Ag(+) and Hg(2+) to 200 microM for Ni(2+)). Exposures of 2 h at these concentrations did not alter cell morphology, staining with trypan blue, or cell number. LPS exposure had no effect on cell number with or without metal ions after 2 h. When metal treatment alone was assessed, none of the metal ions had a significant effect on NF kappa B-DNA binding. However, pretreatment with Co(2+), Ni(2+), Ag(1+), Hg(2+), and Pd(2+) significantly decreased NF kappa B-DNA binding by 40-70% versus LPS alone. Only Cu(2+) had no effect on LPS-induced NF kappa B-DNA complex formation. Pd(2+) lowered, but did not abolish, IL1 beta secretion at concentrations comparable to those that altered NF kappa B-DNA binding. These results suggest that many commonly used metals alter monocyte function at concentrations that are not overtly toxic, and that protein levels controlled in part by NF kappa B also may be altered.

Crystal Structure of Dissimilatory Sulfite Reductase D (DsrD) Protein—Possible Interaction with B- and Z-DNA by Its Winged-Helix Motif

The crystal structure of DsrD from Desulfovibrio vulgaris Hildenborough has been determined at 1.2 Å resolution. DsrD is in a dimeric form in the crystal, and five sulfate anions were located on the surface. The structure of DsrD comprises a winged-helix motif, which shows the highest structural homology to similar motifs found in Z-DNA binding proteins and some B-DNA binding proteins. The core structure of the molecule is constructed by intramolecular interactions of hydrophobic residues, which are well conserved in DNA binding proteins, suggesting that these proteins belong to the same superfamily on the basis of the structure. These results indicate a possible role of DsrD in transcription or translation of genes for enzymes catalyzing dissimilatory sulfite reduction.

Osteopontin Induces Nuclear Factor κB-mediated Promatrix Metalloproteinase-2 Activation through IκBα/IKK Signaling Pathways, and Curcumin (Diferulolylmethane) Down-regulates These Pathways

We have recently reported that osteopontin (OPN) stimulates tumor growth and activation of promatrix metalloproteinase-2 (pro-MMP-2) through nuclear factor kappa B (NF kappa B)-mediated induction of membrane type 1 matrix metalloproteinase (MT1-MMP) in murine melanoma cells (Philip, S., Bulbule, A., and Kundu, G. C. (2001) J. Biol. Chem. 276, 44926-44935). However, the molecular mechanism by which OPN activates NF kappa B and regulates pro-MMP-2 activation in murine melanoma (B16F10) cells is not well defined. We also investigated the mechanism of action of curcumin (diferulolylmethane) on OPN-induced NF kappa B-mediated activation of pro-MMP-2 in B16F10 cells. Here we report that OPN induces phosphorylation and degradation of the inhibitor of nuclear factor kappa B (I kappa B alpha) by inducing the activity of I kappa B kinase (IKK) in these cells. OPN also induces the nuclear accumulation of NF kappa B p65, NF kappa B-DNA binding, and transactivation. However, curcumin a known anti-inflammatory and anticarcinogenic agent suppressed OPN-induced I kappa B alpha phosphorylation and degradation by inhibiting the IKK activity. Moreover, our data revealed that curcumin inhibited the OPN-induced translocation of p65, NF kappa B-DNA binding, and NF kappa B transcriptional activity. The OPN-induced pro-MMP-2 activation and MT1-MMP expression were also drastically reduced by curcumin. Curcumin also inhibited OPN-induced cell proliferation, cell migration, extracellular matrix invasion, and synergistically induced apoptotic morphology with OPN in these cells. Most importantly, curcumin suppressed the OPN-induced tumor growth in nude mice, and the levels of pro-MMP-2 expression and activation in OPN-induced tumor were inhibited by curcumin. To our knowledge, this is the first report that OPN induces NF kappa B activity through phosphorylation and degradation of I kappa B alpha by activating IKK that ultimately triggers the activation of pro-MMP-2 and further demonstrates that curcumin potently suppresses OPN-induced cell migration, tumor growth, and NF kappa B-mediated pro-MMP-2 activation by blocking the IKK/I kappa B alpha signaling pathways.

Proteinase-activated Receptor-2-mediated Activation of Stress-activated Protein Kinases and Inhibitory κB Kinases in NCTC 2544 Keratinocytes

In this study we examined the regulation of the stress-activated protein (SAP) kinases and inhibitory kappa B kinases (IKKs) through stimulation of the novel G-protein-coupled receptor proteinase-activated receptor-2 in the human keratinocyte cell line NCTC2544. Trypsin and the peptide SLIGKV stimulated a time-dependent increase in both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activity. Trypsin also stimulated NF kappa B-DNA binding and the activation of the upstream kinases IKK alpha and -beta. Phorbol 12-myristate 13-acetate also strongly activated both SAP kinases and IKK isoforms, suggesting the potential for a protein kinase C-mediated regulatory mechanism underlying the effects of trypsin. Pre-incubation with selective protein kinase C (PKC) inhibitors GF109203X and Gö6983, or transfection of dominant negative (DN)-PKC alpha, abolished phorbol 12-myristate 13-acetate-mediated c-Jun N-terminal kinase activity, although it only partially inhibited the response to trypsin. In contrast, Gö6983 reduced trypsin-stimulated p38 mitogen-activated protein kinase activity to a greater extent than GF109203X, although DN-PKC alpha or PKC zeta had no substantial effect. Additionally, inhibitors of PKC partially reduced trypsin-stimulated IKK alpha activity but abolished that of IKK beta, whereas DN-PKC alpha but not DN-PKC zeta substantially reduced trypsin-stimulated Flag-IKK beta activity. This study shows for the first time proteinase-activated receptor-2-mediated stimulation of both SAP kinase and IKK signaling and differing roles for PKC isoforms in the regulation of each pathway.

B-DNA Binding with Cobalt(III) and Vanadyl(2+) Derivatives of Tetracationic 5,10,15,20-Tetrakis(4-N-methylpyridyl)porphine: Combined CD, Optical, and Electronic MCD Spectra.

Electronic MCD is used for the first time to obtain electronic and geometric structural insight into the free and DNA-bound states of the six-coordinate CoIII and VO2+ derivatives of 5,10,15,20-tetrakis(4-N-methylpyridyl)porphine. Correlations between MCD, CD, and optical spectra measured in the porphyrin Soret band (or B0) region, and FLDr literature data, are consistent with each metalloporphyrin having a face-on orientation in the major groove of CT DNA, sites 5‘AT3‘ and 5‘CG3‘ being preferred, and with porphyrin electric dipole transition moments (edtms) μexD (most perturbed direction in the bound porphyrin) and μexD (least perturbed) having orientation angles of α/β/β‘ = ∼45°/180°/90° and ∼45°/180°/270°, respectively.

I kappa B alpha degradation is not a requirement for the X-ray-induced activation of nuclear factor kappa B in normal rat astrocytes and human brain tumour cells

Purpose: To investigate the mechanism of NF kappa B activation by X-rays in normal primary rat astrocytes. Materials and methods: Primary cultures of type I astrocytes generated from the cortex of neonatal rats were exposed to X-rays with and without various kinase inhibitors and a protease inhibitor. The nuclear or cytoplasmic protein extracts were collected at specified times after treatment and analysed for NF kappa B-DNA binding activity and I kappa B protein levels. Results: The NF kappa B-DNA binding activity was induced by X-rays in a dose- and time-dependent manner in the absence of I kappa B protein degradation in astrocytes as well as in the human glioma cell line U-373MG. Whereas a protease inhibitor (calpain inhibitor 1) and a protein kinase C inhibitor (CGP-41251) did not affect X-ray-induced NF kappa B-DNA binding, treatment of astrocytes with the tyrosine kinase inhibitor (erbstatin) completely prevented the increase in NF kappa B activity after irradiation. Erbstatin also reduced the phosphorylation of I kappa B alpha after X-ray exposure. Conclusions: These results indicate that, in contrast with the more frequently investigated activators of NF kappa B, radiation-induced activation of this transcription factor proceeds in the absence of I kappa B alpha degradation and requires tyrosine phosphorylation.

An N-terminally truncated third progesterone receptor protein, PR C, forms heterodimers with PR B but interferes in PR B-DNA binding

Multiple progesterone receptor (PR) isoforms may explain in part the complex and diverse biological actions of progestins. Recent studies demonstrate that the human 116 kDa B-receptor (PR B) and the 94 kDa A-receptor (PR A) can have very different transcriptional functions that are cell- and promoter-specific. Additionally, we have shown the existence of a smaller N-terminally truncated 60 kDa progestin-specific binding protein, called the C-receptor (PR C), that has unique transcriptional potentiating properties. In the presence of the other two PR isoforms, PR C enhances the transcriptional activities of the larger PR proteins. In order to determine the mechanism of action for the transcriptional promoting abilities of PR C, the structural and functional properties of PR C were analysed and compared to those of PR A and PR B. PR C consistently displayed a dissociation constant (K d) ∼5 times higher than that for PR B and PR A, suggesting that the N-terminal truncation of PR C results in a conformation different from the two larger PR isoforms, that affects the hormone-binding region and its interaction with hormone. Despite this change, PR C is still capable of forming heterodimers with the larger PR B in solution, as determined by co-immunoprecipitation studies, but PR C interferes in tight PR B binding to DNA in gel-shift assays. Surprisingly, progestin and anti-progestin autoregulation of PR C protein levels parallel those for PR B and PR A.

Redox regulation of manganese superoxide dismutase.

The importance of reactive oxygen species (ROS) or changes in cellular redox state in signal transduction and gene regulation is becoming increasingly evident. In this study, we tested the hypothesis that ROS are directly involved in the induction of the mitochondrial antioxidant manganese superoxide dismutase (MnSOD) and mediate the induction of MnSOD by tumor necrosis factor-alpha (TNF-alpha). Pretreatment of human pulmonary adenocarcinoma cells H441 with the antioxidants N-acetyl-L-cysteine (NAC) and nordihydroguaiaretic acid (NDGA) blocked MnSOD induction by TNF-alpha, implicating ROS as a signaling agent in this pathway. Treatment of H441 cells with the exogenous oxidants hydrogen peroxide (H2O2) and diamide increased MnSOD mRNA, supporting the hypothesis that ROS directly affect expression of MnSOD. The temporal pattern of MnSOD induction differed for TNF-alpha and H2O2, suggesting distinct signaling pathways. DNA binding of two redox-sensitive transcription factors, NF-kappa B and activator protein (AP)-1, was evaluated. TNF-alpha increased nuclear factor (NF)-kappa B-DNA binding, an effect blocked by pretreatment with NAC. H2O2 did not alter NF-kappa B-DNA binding. There was no evidence of AP-1 binding in cells treated with either TNF-alpha or H2O2. We conclude that ROS directly alter MnSOD expression and are involved in the induction of MnSOD by TNF-alpha.

Heavy chain dominance in the binding of DNA by a lupus mouse monoclonal autoantibody

Antibodies H241 and 2C10 are lupus mouse IgG autoantibodies that bind native DNA. In previous experiments, oligonucleotide antigens affinity-labeled both H and L chains of H241 but only the H chain of antibody 2C10. Primary structures of the V regions of the 2C10 H and L chains and the H241 L chain, determined from cDNA, help to explain the previous affinity-labeling experiments. The 2C10 L chain CDRs had several Asp residues and a net negative charge of five, whereas the 2C10 H chain CDRs had four Arg residues and a net positive charge of five. The L chain CDRs of H241 had a net positive charge of one. [The H241 H chain cDNA sequence was published previously by Gangemi et al. (1993) J. Immun. 151, 4660–4671]. Plasmid vectors were used for bacterial expression of H and L chains of 2C10 alone and in combinations in single chain Fv (scFv) molecules. The H chain alone bound native DNA as well as or better than the H-plus-L chain scFv. The H chain alone also bound Z-DNA. Combination of the 2C10 H chain with the L chain of an anti-Z-DNA antibody maintained the selectivity for Z-DNA, whereas its combination with the 2C10 L chain (in the 2C10 Fab) yielded selective B-DNA binding. The results with 2C10 match other examples in which the H chain is sufficient for DNA binding but selectivity is modulated by the L chain. The H chain binding to autoantigen may reflect selective events in early stages of B cell development.

Rhinovirus stimulation of interleukin-6 in vivo and in vitro. Evidence for nuclear factor kappa B-dependent transcriptional activation.

To further understand the biology of rhinovirus (RV), we determined whether IL-6 was produced during RV infections and characterized the mechanism by which RV stimulates lung cell IL-6 production. In contrast to normals and minimally symptomatic volunteers, IL-6 was detected in the nasal washings from patients who developed colds after RV challenge. RV14 and RV1A, major and minor receptor group RVs, respectively, were potent stimulators of IL-6 protein production in vitro. These effects were associated with significant increases in IL-6 mRNA accumulation and gene transcription. RV was also a potent stimulator of IL-6 promoter-driven luciferase activity. This stimulation was modestly decreased by mutation of the nuclear factor (NF)-IL-6 site and abrogated by mutation of the NF-kappa B site in this promoter. An NF-kappa B-DNA binding activity, mediated by p65, p50, and p52 NF-kappa B moieties, was rapidly induced in RV-infected cells. Activator protein 1-DNA binding was not similarly altered. These studies demonstrate that IL-6 is produced during symptomatic RV infections, that RVs are potent stimulators of IL-6 elaboration, and that RV stimulation IL-6 production is mediated by an NF-kappa B-dependent transcriptional stimulation pathway. IL-6 may play an important role in the pathogenesis of RV infection, and NF-kappa B activation is likely to be an important event in RV-induced pathologies.

A demonstration of the intrinsic importance of stabilizing hydrophobic binding and non-convalent van der waals contacts dominant in the non-covalent CC-1065/B-DNA binding

The comparative DNA binding properties and cytotoxic activity of CDPI n methyl esters ( n = 1–5) vs. PDE-I n methyl esters ( n = 1–3) are detailed in studies which provide experimental evidence for the intrinsic importance of stabilizing hydrophobic binding and non-covalent van der Waals contacts dominant in the CC-1065/B-DNA minor groove binding. High affinity minor groove binding to DNA was established through: (1) the observation of CDPI 3 binding (UV) but not unwinding of supercoiled DNA (φ174 RFI DNA) thus excluding intercalative binding; (2) the observation of CDPI 3 binding to T 4 phage DNA (UV, ΔT m) in which the major groove is occluded by glycosylation thus excluding major groove binding; (3) the observation of salt (Na +) concentration independent high affinity CDPI 3 binding to poly(dA) · poly(dT) thus excluding simple electrostatic binding to the DNA phosphate backbone; and further inferred through (4) the observation of an intense induced dichroism (ICD, poly(dA) · poly(dT) and poly(dG) · poly(dC) [ φ] 358 23 = 24 000 and 23 500). This high affinity minor groove binding is sufficient to produce a potent cytotoxic effect. From thermal denaturation studies and relative binding constant determinations it was demonstrated that: (1) CDPI 3 represents the optimum binding unit (25°C) within the oligomeric CDPI n agents studied (CDPI 3 > CDPI 5 ≅ CDPI 4 ⩾ CDPI 2 > CDPI 1); (2) CDPI 3 minor groove binding spans five base-pairs or one-half a helix turn of double-stranded DNA which represents the largest accessible binding site available for synchronous binding of the rigid agent termini [poly(dA) · poly(dT) and poly[d(A-T) · poly[d(A-T)] agent/base-pair ratio = 0.2]; (3) partial bound forms of the larger oligomer agents, i.e CDPI 2-bound CDPI 4, constitute productive and relevant DNA/agent complexes; (4) the PDE-I 3/DNA complexes are thermodynamically more stable and kinetically more labile than the corresponding CDPI n /DNA complexes; (5) the qualitative and relative quanlitative free energies of binding of the agents to DNA follows trends depicted in a quantitative molecular modeling study from which estimates of the gas phase, relative enthalpies binding were available (poly(dA) · poly(dT) and poly[d(A-T)] · poly[d(A-T)] > poly(dG) · poly(dC)/PDE-I 3 > CDPI 3 > PDE-I 2 > CDPI 2-bound CDPI 4 > CDPI 2 > CDPI 1); (6) the absence of CDPI n functionality that would permit stabilization of DNA/CDPI n complexes through hydrogen bonding or substantial stabilizing electrostatic interactions suggests that the binding affinity of the agents is derived principally if not exclusively from hydrophobic binding and stabilizing van der Waals contacts (affinity = hydrophobic binding); (7) the CDPI n agents exhibit a substantial preference of A · T rich vs. G · C rich DNA minor groove binding [CDPI n: K b [poly(dA) · poly(dT)], poly[d(A-T)] · poly[d(A-T)] > K b[poly(dG) · poly(dC)]; CDPI 3 ( frsol|(25°C)/ ΔΔG° poly(dA) · poly(dT) vs. poly(dG) · poly(dC) = −2.6 kcal] which represents preferential binding to minor groove regions in which stabilizing agent/DNA van der Waals contacts would be enhanced with the deeper penetration of the agents into the narrower A · T rich DNA minor groove (specificity = accessible hydrophobic binding); and (8) the trends in the cytotoxic potency of non-covalent binding agents (CDPI n : CDPI 4 > CDPI 3 ⪢ CDPI 2, CDPI 1; PDE-I n :PDE-I 3 > PDE-I 2; CDPI n /PDE-I n :PDE-I 2 > CDPI 2, PDE-I 3 > CDPI 3) follow the trends depicted for full structure binding of the agents. Consequently, the studies suggest that the antitumor antibiotic ( + )-CC-1065 is best represented as a selective alkylating group superimposed upon the rigid CDPI 3 skeleton rather than as reactive alkylating agent (CPI) attached to a DNA binding agent (PDE-I 2). The initial non-covalent DNA binding of the agent may be expected to exhibit a substantial preference for A · T rich DNA minor groove binding that may be attributed to accessible hydrophobic binding and is optimal with the properties and size of the rigid, hydrophobic trimer (CDPI 3/PDE-I 3).

Interaction of synthetic analogues of distamycin and netropsin with nucleic acids. Does curvature of ligand play a role in distamycin-DNA interactions?

Distamycin and netropsin, a class of minor groove binding nonintercalating agents, are characterized by their B-DNA and A-T base-specific interactions. To understand the conformational and chemical basis of the above specificities, the DNA-binding characteristics of a novel synthetic analogue of distamycin have been studied. The analogue, mPD derivative, has the requisite charged end groups and a number of potential hydrogen-bonding loci equal to those of distamycin. The difference in the backbone curvatures of the ligands, distamycin, the mPD derivative, and NSC 101327 (another structurally analogous compound), is a major difference between these ligands. UV and CD spectroscopic studies reported here show the following salient features: The mPD derivative recognizes only B-DNA, to which it binds via the minor groove. On the other hand, unlike distamycin, it binds with comparable affinities to A-T and G-C base pairs in a natural DNA. These DNA-binding properties are compared with those reported earlier for distamycin and NSC 101327 [Zimmer, Ch., & Wahnert, U. (1986) Prog. Biophys. Mol. Biol. 47, 31-112]. The backbone structures of these three ligands were compared to show the progressive decrease in curvatures in the order distamycin, mPD derivative, and NSC 101327. The plausible significance of the backbone curvature vis-à-vis the characteristic B-DNA and AT-specific binding of distamycin is discussed. To our knowledge, this is the first attempt (with a model synthetic analogue) to probe the possible influence of backbone curvature upon the specificity of interactions of the distamycin class of groove-binding ligands with DNA.