Divalent DNA Research Articles

Influence of the N-Terminal Domain and Divalent Cations on Self-Association and DNA Binding by the Saccharomyces cerevisiae TATA Binding Protein

The localization of a single tryptophan to the N-terminal domain and six tyrosines to the C-terminal domain of TBP allows intrinsic fluorescence to separately report on the structures and dynamics of the full-length TATA binding protein (TBP) of Saccharomyces cerevisiae and its C-terminal DNA binding domain (TBPc) as a function of self-association and DNA binding. TBPc is more compact than the C-terminal domain within the full-length protein. Quenching of the intrinsic fluorescence by DNA and external dynamic quenchers shows that the observed tyrosine fluorescence is due to the four residues surrounding the "DNA binding saddle" of the C-terminal domain. TBP's N-terminal domain unfolds and changes its position relative to the C-terminal domain upon DNA binding. It partially shields the DNA binding saddle in octameric TBP, shifting upon dissociation to monomers to expose the saddle to DNA. Structure-energetic correlations were obtained by comparing the contribution that electrostatic interactions make to DNA binding by TBP and TBPc; DNA binding by TBPc is more hydrophobic than that by TBP, suggesting that the N-terminal domain either interacts with bound DNA directly or screens a part of the C-terminal domain, diminishing its electronegativity. The competition between divalent cations, K+, and DNA is not straightforward. Divalent cations strengthen binding of TBP to DNA and do so more strongly for TBPc. We suggest that divalent cations affect the structure of the bound DNA perhaps by stabilizing its distorted conformation in complexes with TBPc and TBP and that the N-terminal domain mimics the effects of divalent cations. These data support an autoinhibitory mechanism in which competition between the N-terminal domain and DNA for the saddle diminishes the DNA binding affinity of the full-length protein.

Molecular dynamics in calf‐thymus DNA, at neutral and low pH, in the presence of Na+, Ca2+ and Mg2+ ions: A Raman microspectroscopic study

In this paper the Raman total half bandwidths of calf‐thymus DNA vibrations have been measured as a function of pH, monovalent and divalent cations′ type and concentration. The dependence of different band parameters on DNA molecular subgroup structure, on pH and on Na+, Ca2+ and Mg2+ ions concentrations, respectively, are reported. It is shown that changes in (sub)picosecond dynamics of molecular subgroups in calf‐thymus DNA can be monitored with confocal Raman microspectroscopy.The half bandwidths and the global relaxation times for the vibrations at 728 cm−1 (dA), 785 cm−1 (dC), 1094 cm−1 (PO2−), 1377 cm−1 (dA, dG, dT, dC), 1488 cm−1 (dG, dA) and 1580 cm−1 (dG, dA) of calf‐thymus DNA are presented. The full‐widths at half‐height (FWHH) of the bands in calf‐thymus DNA are typically in the wavenumber range from 7.4 to 31 cm−1. The bandwidths in the Raman spectra are sensitive to a dynamics active on a time scale from 0.34 to 1.44 ps.Low pH‐induced melting of double helical structure in calf‐thymus DNA results for some bands in shorter global relaxation times, as a consequence of the increased interaction of the base moieties with the solvent molecules.The molecular dynamics characterizing the 785, 1094, 1377 and 1580 cm−1 vibrations, is faster in the case of high divalent cations DNA sample (pH 7), as compared to the respective low divalent cations DNA sample (pH 7), for both Ca2+ and Mg2+ ions. The vibrational energy transfer process of the guanine band at 1488 cm−1 is slower for the high salt DNA sample, pH 7 as compared to the corresponding low salt DNA sample, pH 7, for both Ca2+ and Mg2+. Molecular dynamics characterizing the vibration at 1488 cm−1 is faster for DNA sample at high Na+ ions (pH 7), as compared to the DNA sample at low Na+ ions (pH 7).As far as the CaDNA and MgDNA complexes are concerned (pH 7), the global relaxation times of some base vibrations decrease for the case of magnesium ions, as compared to the case of the same concentration of calcium ions. The different ionic radius of the two types of metal cations (0.72 Å for Mg and 0.99 Å for Ca) were considered in explaining these results.Molecular relaxation processes of DNA subgroups, upon lowering the pH, in the presence of Na+, Ca2+ and Mg2+ ions are presented. Particularly, at low Ca2+ concentration, upon lowering the pH, the molecular dynamics of DNA subgroups corresponding to vibrations at 728, 1376, 1488 and 1580 cm−1 is much faster, probably due to the denaturation process of the double helical DNA.

Effects of Bridge Ions, DNA Species, and Developing Temperature on Flat-Lying DNA Monolayers

Recently, we have successfully constructed flat-lying DNA monolayers on a mica surface (J. Phys. Chem. B 2006, 110, 10792-10798). In this work, the effects of various factors including bridge ions, DNA species, and developing temperature on the configuration of DNA monolayers have been investigated by atomic force microscopy (AFM) in detail. AFM results show that the species of bridge ions and developing temperature play a crucial role during the formation process. For example, the divalent cation Zn2+ resulted in many DNA chains stuck side by side in the monolayers due to the strong interactions between it and DNA's bases or the mica surface. Most DNA chain's conglutinations disappeared when the developing temperature was higher than 40 degrees C. Cd2+ and Ca2+ produced more compact DNA monolayers with some obvious aggregations, especially for the DNA monolayers constructed by using Ca2+ as the bridge ion. Co2+ produced well-ordered, flat-lying DNA monolayers similar to that of Mg2+. Furthermore, it was found that the flat-lying DNA monolayers could still form on a mica surface when plasmid DNA pBR 322 and linear DNA pBR 322/Pst I were used as the DNA source. Whereas, it was hard to form DNA monolayers on a (3-aminopropyl)triethoxysilane-mica surface because the strong interactions between DNA and substrate prevented the lateral movement of DNA molecules. These results suggested that the appropriate interactions between divalent cations and DNA or mica surface were important for the formation of flat-lying DNA monolayers. The obtained information is a necessary supplement to our previous studies on the formation kinetics of such monolayers and may be useful for practical application of the monolayers and further theoretical studies.

The structural diversity of DNA–neutral phospholipids–divalent metal cations aggregates: a small-angle synchrotron X-ray diffraction study

We investigate the structure of aggregates formed due to DNA interaction with saturated neutral phosphatidylcholines [dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine] in presence of Ca(2+) and Mg(2+) cations using simultaneous synchrotron small- and wide-angle X-ray diffractions. For DPPC:DNA = 3:1 mol/base and in the range of 1-50 mM Ca(2+), the diffractograms show structural heterogeneity of aggregates. We observe the coexistence of two lamellar phases in aggregates prepared at 1 mM Ca(2+): L(x) phase with the DNA strands (of unknown organization) intercalated in water layers between adjacent lipid bilayers and L(DPPC) phase of DPPC bilayers without any divalent cations and DNA strands. Aggregates prepared in the range 2-50 mM Ca(2+) show a condensed gel lamellar phase L (g) (c) with the lipid bilayer periodicity d approximately 8.0 nm, and the DNA-DNA interhelical distance d (DNA) approximately 5.1 nm. The increase of temperature induces the decrease in the intensity and the increase in the width of the DNA related peak. In the fluid state, the condensed lamellar phase L (alpha) (c) gradually converts into L(x) phase. The aggregates do not exhibit rippled P(beta) phase. The thermal behaviour of aggregates was investigated in the range 20-80 degrees C. Applying heating-cooling cycles, the aggregates converted into energetically more favourable structure: a condensed lamellar phase L(c) (or L(x)) is preserved or we observe lateral segregation of the DNA strands and metal cations (L(x) phase) in coexistence with L(PC) phase of pure phospholipids.

Protective Immunity Elicited by a Divalent DNA Vaccine Encoding Both the L7/L12 and Omp16 Genes ofBrucella abortusin BALB/c Mice

This study was designed to evaluate the immunogenicity and the protective efficacy of a divalent fusion DNA vaccine encoding both the Brucella abortus L7/L12 protein (ribosomal protein) and Omp16 protein (outer membrane lipoprotein), designated pcDNA3.1-L7/L12-Omp16. Intramuscular injection of this divalent DNA vaccine into BALB/c mice elicited markedly both humoral and cellular immune responses. The specific antibodies exhibited a dominance of immunoglobulin G2a (IgG2a) over IgG1. In addition, the dual-gene DNA vaccine elicited a strong T-cell proliferative response and induced a large amount of gamma interferon-producing T cells upon restimulation in vitro with recombinant fusion protein L7/L12-Omp16, suggesting the induction of a typical T-helper-1-dominated immune response in vivo. This divalent DNA vaccine could also induce a significant level of protection against challenge with the virulent strain B. abortus 544 in BALB/c mice. Furthermore, the protection level induced by the divalent DNA vaccine was significantly higher than that induced by the univalent DNA vaccines pcDNA3.1-L7/L12 or pcDNA3.1-Omp16. Taken together, the results of this study verify for the first time that the Omp16 gene can be a candidate target for a DNA vaccine against brucellosis. Additionally, a divalent genetic vaccine based on the L7/L12 and Omp16 genes can elicit a stronger cellular immune response and better immunoprotection than the relevant univalent vaccines can.

Comparing mono- and divalent DNA groove binding cyanine dyes—Binding geometries, dissociation rates, and fluorescence properties

The unsymmetrical cyanine dyes BOXTO-PRO and BOXTO-MEE were derived from the DNA groove binder BOXTO, by adding a positively charged or a non-ionic hydrophilic tail to BOXTO, respectively. The main objective was to obtain more efficient DNA probes, for instance in electrophoresis and microscopy, by slowing down the dissociation of BOXTO from DNA. The interactions with mixed sequence DNA was studied with fluorescence and absorbance spectroscopy, stopped-flow dissociation and gel electrophoresis. Both the derivatives are groove bound as BOXTO, and have similar fluorescence properties when bound to mixed sequence DNA in free solution. BOXTO-PRO exhibits a slower dissociation than BOXTO from DNA, whereas the dissociation rate for BOXTO-MEE is faster and, unexpectedly independent of the ionic strength. During gel electrophoresis both BOXTO-PRO and BOXTO-MEE exhibit a faster dissociation rate than BOXTO. Still, BOXTO-PRO seems to be a good alternative as DNA probe, especially for applications in free solution where the dissociation is slower than for the corresponding intercalator TOPRO-1.

The structure of DNA–DOPC aggregates formed in presence of calcium and magnesium ions: A small-angle synchrotron X-ray diffraction study

The structure of aggregates formed due to DNA interaction with dioleoylphosphatidylcholine (DOPC) vesicles in presence of Ca 2+ and Mg 2+ cations was investigated using synchrotron small-angle X-ray diffraction. For DOPC/DNA = 1:1 mol/base and in the range of concentration of the cation 2+ 0–76.5 mM, the diffractograms show the coexistence of two lamellar phases: L x phase with repeat distance d Lx ∼ 8.26–7.39 nm identified as a phase where the DNA strands are intercalated in water layers between adjacent lipid bilayers, and L DOPC phase with repeat distance d DOPC ∼ 6.45–5.65 nm identified as a phase of partially dehydrated DOPC bilayers without any divalent cations and DNA strands. The coexistence of these phases was investigated as a function of DOPC/DNA molar ratio, length of DNA fragments and temperature. If the amount of lipid increases, the fraction of partially dehydrated L DOPC phase is limited, depends on the portion of DNA in the sample and also on the length of DNA fragments. Thermal behaviour of DOPC + DNA + Ca 2+ aggregates was investigated in the range 20–80 °C. The transversal thermal expansivities of both phases were evaluated.

Immunization with a dicistronic plasmid expressing a truncated form of bovine herpesvirus-1 glycoprotein D and the amino-terminal subunit of glycoprotein B results in reduced gB-specific immune responses

As an approach to create a divalent DNA vaccine, a truncated secreted version of bovine herpesvirus-1 (BHV-1) glycoprotein D (tgD) and the amino-terminal subunit of glycoprotein B (gBb) were expressed from a dicistronic plasmid, designated pSLIAtgD-IRES-gBb. Intradermal immunization of mice with pSLIAtgD-IRES-gBb or a mixture of plasmids encoding tgD (pSLIAtgD) and gBb (pSLIAgBb) by needle injection or gene gun elicited strong tgD-specific immune responses. However, a significant reduction in gBb-specific immune responses was observed upon immunization of mice with pSLIAtgD-IRES-gBb or a mixture of pSLIAtgD and pSLIAgBb in comparison to immunization with pSLIAgBb alone. This reduction in gBb-specific immune responses induced by pSLIAtgD-IRES-gBb was due to production of low amounts of gBb from pSLIAtgD-IRES-gBb, inefficient processing and transport of gBb, and possibly competition for antigen-presenting cells by tgD and gBb. These results indicate that, although divalent plasmids may be used to express different antigens, the efficacy of vaccination with such plasmids may be influenced by the plasmid design and the characteristics of the expressed antigens.

The divalent cation-induced DNA condensation studied by atomic force microscopy and spectra analysis

Three types of DNA condensates in the presence of divalent metal ion (Mg, Mn and Cu) and EB simultaneously have been observed by atomic force microscopy (AFM). In the presence of Mg (II) and EB, DNA molecular exhibits three or four maggot-like branch crossing structures at the same point, which were constructed by some bead-like particles in each branch. However, in the presence of Cu (II) or Mn (II)–EB, DNA molecular forms a network or a semicircular structure, respectively. Their average height was 0.95±0.5 nm (Cu (II)–DNA–EB), 17±5 nm (Mg (II)–DNA–EB) and 12±3 nm (Mn (II)–DNA–EB), respectively. However, DNA molecular or DNA–EB complex still remained a line double-stranded structure. The fluorescence, melting point and CD spectra indicate that the different morphologies induced by divalent metal ion (Mg (II), and Mn (II) and Cu (II)) are related to its binding position in molecular DNA.

The Structure of a CREB bZIP·Somatostatin CRE Complex Reveals the Basis for Selective Dimerization and Divalent Cation-enhanced DNA Binding

The cAMP responsive element-binding protein (CREB) is central to second messenger regulated transcription. To elucidate the structural mechanisms of DNA binding and selective dimerization of CREB, we determined to 3.0 A resolution, the structure of the CREB bZIP (residues 283-341) bound to a 21-base pair deoxynucleotide that encompasses the canonical 8-base pair somatostatin cAMP response element (SSCRE). The CREB dimer is stabilized in part by ionic interactions from Arg(314) to Glu(319') and Glu(328) to Lys(333') as well as a hydrogen bond network that links the carboxamide side chains of Gln(322')-Asn(321)-Asn(321')-Gln(322). Critical to family selective dimerization are intersubunit hydrogen bonds between basic region residue Tyr(307) and leucine zipper residue Glu(312), which are conserved in all CREB/CREM/ATF-1 family members. Strikingly, the structure reveals a hexahydrated Mg(2+) ion bound in the cavity between the basic region and SSCRE that makes a water-mediated DNA contact. DNA binding studies demonstrate that Mg(2+) ions enhance CREB bZIP:SSCRE binding by more than 25-fold and suggest a possible physiological role for this ion in somatostatin cAMP response element and potentially other CRE-mediated gene expression.

M-DNA: a complex between divalent metal ions and DNA which behaves as a molecular wire

M-DNA is a complex of DNA with divalent metal ions (Zn 2+, Co 2+, or Ni 2+) which forms at pH conditions above 8. Upon addition of these metal ions to B-DNA at pH 8.5, the pH decreases such that one proton is released per base-pair per metal ion. Together with previous NMR data, this result demonstrated that the imino proton in each base-pair of the duplex was substituted by a metal ion and that M-DNA might possess unusual conductive properties. Duplexes of 20 base-pairs were constructed with fluorescein (donor) at one end and rhodamine (acceptor) at the other. Upon formation of M-DNA (with Zn 2+) the fluorescence of the donor was 95 % quenched. Fluorescence lifetime measurements showed the presence of a very fast component in the decay kinetics with τ⩽ 10 ps. The fast component was absent in B-DNA and in M-DNA lacking an acceptor chromophore; a result which is only consistent with electron transfer. Efficient signal transduction was also observed between the two fluorophores separated by 54 base-pairs (over 150 Å) in an M-DNA duplex. The addition of a sequence-specific DNA-binding protein prevented the flow of electrons and this was reversed by protease digestion. Therefore, M-DNA behaves as a molecular wire and could be manipulated to prepare self-assembling electronic circuits.

Substrate-Length-Dependent Activities of Human Immunodeficiency Virus Type 1 Integrase in Vitro: Differential DNA Binding Affinities Associated with Different Lengths of Substrates

Human immunodeficiency virus type 1 integrase (HIV-IN) is an enzyme essential for the integration of viral DNA into the host chromosome, a process that is an attractive target for drug development. In vitro assays have been developed to study both components of the integration process, the 3'-processing and strand transfer reactions. However, major discrepancies between results obtained from in vivo and in vitro events raise concerns as to the biological relevance of activities observed in vitro. These discrepancies include the size of the substrate and the nature of the divalent cation used. In this study, we characterized activities of HIV-IN with oligonucleotide substrates varying in length. Our previous studies indicate that the preferred cation in vitro for 3'-processing is altered from Mn2+ to Mg2+ by increasing the length of the oligonucleotide substrate. This study demonstrates that HIV-IN efficiently catalyzes Mg(2+)-dependent 3'-processing while repressing the strand transfer reaction. Substrate competition studies indicate that longer substrates preferentially bind to the viral DNA binding site of the integrase, whereas the shorter substrate has much less specificity. In addition, the shorter substrate requires a higher concentration of Mg2+, indicating that there is an alteration in the metal binding affinity associated with the varying substrates. Our results show that substrate-length-dependent differential activities are due to differences in the divalent metal binding and DNA binding affinities associated with the different substrates. These results suggest that the structure of the viral DNA is an important factor in differentiating the donor and target substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism and optimized conditions for PEG mediated DNA transfection into plant protoplasts

Experimental conditions influencing DNA uptake efficiency by maize protoplasts in polyethyleneglycol (PEG) mediated transfection experiments have been studied systematically. The data provide evidence that the extracellular DNA is precipitated efficiently by combined action of PEG together with divalent cations and DNA is taken up by the plant protoplasts in the precipitated form. The particle size is strongly effected by the pH of the PEG solution. At optimal pH 6- 6.5 a very fine and homogenous precipitate forms in presence of Ca(2+) and Mg(2+) ions and is efficiently incorporated by maize and rice protoplasts.

Aspects of polymerization catalyzed by RNA polymerase from Pseudomonas indigofera

1. 1. RNA polymerase (nucleoside triphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) from Pseudomonas indigofera has been purified 135-fold to virtual homogeneity. It can be recovered in 25% yield. Optimal synthesis of riboheteropolymer occurs at 37° and within a pH range of 7.6–9.0. It requires the presence of the four nucleoside triphosphates, 5 mM divalent metallic ion and DNA. The synthesis is stimulated by spermidine and inhibited by actinomycin D. The data suggest that product inhibition occurs. In the absence of DNA, heteropolymer is optimally made at five-fold lower Mg 2+ concentration and it is either adenylate rich or contains frequent runs of adenylate residues. 2. 2. If input is restricted to ATP or UTP, polyadenylate or polyuridylate is synthesized in the absence of exogenous DNA and still lower concentrations of Mg 2+ suffice. These observations suggest that ribohomopolymer synthesis is an inherent property of DNA-primed RNA polymerase.