Photophysics of ethidium bromide complexed to ct-DNA: a maximum entropy study

Time-integrated and time-resolved fluorescence spectroscopies have been used to probe the photophysical properties of ethidium bromide (Eb) complexed to calf thymus DNA (ct-DNA). Fluorescence decay profiles are obtained using the technique of time-correlated single photon counting (TCSPC), and subsequently analysed using conventional sum-of-exponential (SOE) routines and also the maximum entropy method (MEM). Through use of these methods and simulated decay data, it is demonstrated that the kinetics of Eb in the presence of ds-DNA are best described by a generic model consisting of three exponential terms. At all DNA:Eb ratios and NaCl concentrations studied, free Eb is detected. Furthermore, Eb is found to interact with ds-DNA through two mechanisms, each distinguishable by its fluorescence decaytime. Eb is shown to interact with DNA through classic intercalation, and also through binding at secondary sites. The component decaytimes are shown to be a function of NaCl concentration but independent of DNA:Eb molar ratio.     

Electrochemical spectroscopic investigations on the interaction of an ytterbium complex with DNA and their analytical applications such as biosensor

Metal ion-DNA interactions are important in nature, often changing the genetic material's structure and function. A new Yb complex of YbCl₃ (tris(8-hydroxyquinoline-5-sulfonic acid) ytterbium) was synthesized and utilized as an electrochemical indicator for the detection of DNA oligonucleotide based on its interaction with Yb(QS)₃. Cyclic voltammetry (CV) and fluorescence spectroscopy were used to investigate the interaction of Yb(QS)₃ with ds-DNA. It was revealed that Yb(QS)₃ presented an excellent electrochemical activity on glassy carbon electrode (GCE) and could intercalate into the double helix of double-stranded DNA (ds-DNA). The binding mechanism of interaction was elucidated on glassy carbon electrode dipped in DNA solution and DNA modified carbon paste electrode by using differential pulse voltammetry and cyclic voltammetry. The binding ratio between this complex and ds-DNA was calculated to be 1:1. The extent of hybridization was evaluated on the basis of the difference between signals of Yb(QS)₃ with probe DNA before and after hybridization with complementary DNA. With this approach, this DNA could be quantified over the range from 1 × 10⁻⁸ to 1.1 × 10⁻⁷ M. The interaction mode between Yb(QS)₃ and DNA was found to be mainly intercalative interaction. These results were confirmed with fluorescence experiments.     

Detection of protein-DNA interaction with a DNA probe: distinction between single-strand and double-strand DNA-protein interaction

A simple, direct method for the detection of DNA-protein interaction was developed with electrochemical methods. Single-stranded DNA (ss-DNA) probes were prepared through the chemical bonding of an oligonucleotide to a polymer film bearing carboxylic acid groups, and double-stranded DNA (ds-DNA) probes were prepared through hybridization of the complementary sequence DNA on the ss-DNA probe. Impedance spectroscopy and differential pulse voltammetry (DPV) distinguished the interaction between the DNA probes with mouse Purbeta (mPurbeta), an ss-DNA binding protein, and with Escherichia coli MutH, a ds-DNA binding protein. Impedance spectra obtained before and after the interaction of DNA probes with these proteins clearly showed the sequence-specific ss-DNA preference of mPurbeta and the sequence-specific ds-DNA preference of MutH. The concentration dependence of proteins on the response of the DNA probes was also investigated, and the detection limits of MutH and mPurbeta were 25 and 3 microg/ml, respectively. To confirm the impedance results, the variation of the current oxidation peak of adenine of the DNA probe was monitored with DPV. The formation constants of the complexes formed between the probe DNA and the proteins were estimated based on the DPV results.     

EB荧光分析法测定肿瘤细胞DNA交联及增殖活性

应用EB荧光分析法测定体外培养人宫颈癌细胞株(HeLa)、人白血病细胞株(HL-60),增殖性和非增殖性人外周血淋巴细胞(PBL)的DNA含量及其交联度(DNA cross-link),并据此研究不同增殖状态细胞与其DNA百分交联度(ct%)的相互关系.结果显示,HeLa细胞、HL-60细胞、增殖性和非增殖性PBL的DNA ct%分别为36.5、 22.5、 20.2和0,表明不同增殖速度或周期的细胞具有不同的DNA交联反应,而非增殖性细胞或G₀期细胞不产生DNA交联反应.     

Nuclear aggregates of polyamines

Nuclear aggregates of polyamines (NAPs) are cyclic supramolecular compounds made of polyamines and phosphate groups. Three different aggregates, s-NAP, m-NAP and l-NAP, with a molecular weight of 1035, 5175 and 9552 Da, respectively, are described. These molecules interact with genomic DNA. In consequence of this interaction, NAPs not only protect DNA from nucleases with extraordinarily greater efficiency than single polyamines (spermine, spermidine and putrescine), but also induce noticeable changes in DNA condensation status, as shown by temperature-dependent modifications of DNA electrophoretic properties. The biochemical characterization of these compounds has allowed the definition of a structural model for each NAP. According to this model, five s-NAPs assemble together to form a m-NAP unit. We hypothesize that the complexation of s-NAP into m-NAP favours the transition to Z-DNA through the progressive widening of DNA strands and the exposure of bases. We propose that NAPs, by wrapping the DNA helixes, form supramolecular tunnel-like structures that confer efficient protection without affecting DNA elasticity.     

The effect of Ca2+ ions on DNA compaction in the complex with non-histone chromosomal protein HMGB1

The analysis of absorption and circular dichroism spectra in UV and IR regions showed that Ca2+ ions interact both with the phosphate groups of DNA and with the HMGB1 protein. Not only negatively charged C-terminal part of the protein molecule participates in interaction with metal ions but also its DNA-binding domains. The latter fact leads to the change of the mode of protein-DNA interaction. The presence of Ca2+ ions prevents formation of ordered supramolecular structures, specific for the HMGB1-DNA complexes, though promotes intermolecular aggregation. The structure of the complexes between DNA and the protein HMGB1 lacking C-terminal tail appears to be the most sensitive to the presence of Ca2+ ions. The data obtained allow to conclude that Ca2+ ions do not play a structural role in the HMGB1/DNA complexes and the presence of these ions is not necessary to DNA compaction in such systems.     

Functional interplay between the Bacillus subtilis DnaD and DnaB proteins essential for initiation and re-initiation of DNA replication

Initiation and re-initiation of chromosomal DNA replication in bacteria rely on divergent multiprotein assemblies, which direct the functional delivery of the replicative helicase on single-stranded DNA (ssDNA) at specific sites. These two processes are triggered either at the single chromosomal origin oriC or at arrested forks by the conserved DnaA and PriA proteins respectively. In Bacillus subtilis, these two pathways further require the three essential proteins DnaB, DnaD and DnaI, restrictively encoded in Gram positive bacteria of low GC content. We have recently shown that DnaI and DnaB act as a pair of loaders of the DnaC replicative helicase. The role of DnaD appeared more enigmatic. It was previously shown to interact with DnaA and to display weak ssDNA binding activity. Here, we report that purified DnaD can interact physically with PriA and with DnaB. We show that the lethality of the temperature-sensitive dnaD23 mutant can be suppressed by different DnaB point mutants, which were found to be identical to the suppressors of priA null mutants. The DnaD23 protein displays lower ssDNA binding activity than DnaD. Conversely, the DnaB75 protein, the main dnaD23 suppressor, has gained affinity for ssDNA. Finally, we observed that this interplay between DnaD and DnaB is crucial for their concerted interaction with SSB-coated ssDNA, which is the expected substrate for the loading of the replicative helicase in vivo. Altogether, these results highlight the need for both DnaD and DnaB to interact individually and together with ssDNA during the early stages of initiation and re-initiation of chromosomal DNA replication. They also point at a main structural role of DnaD in the multiprotein assemblies built during these two essential processes.     

The histone-like protein HU binds specifically to DNA recombination and repair intermediates

The heterodimeric HU protein associated with the Escherichia coli nucleoid shares some properties with histones and HMG proteins. HU binds DNA junctions and DNA containing a nick much more avidly than double-stranded (ds-) DNA. Cells lacking HU are extremely sensitive to gamma irradiation and we wondered how HU could play a role in maintaining the integrity of the bacterial chromosome. We show that HU binds with high affinity to DNA repair and recombination intermediates, including DNA invasions, DNA overhangs and DNA forks. The DNA structural motif that HU specifically recognizes in all these structures consists of a ds-DNA module joined to a second module containing either ds- or single-stranded (ss-) DNA. The two modules rotate freely relative to one another. Binding specificity results from the simultaneous interaction of HU with these two modules: HU arms bind the ds-DNA module whereas the HU body contacts the 'variable' module containing either ds- or ss-DNA. Both structural motifs are recognized by HU at least 1000-fold more avidly than duplex DNA.     

Iminothiol/thiourea tautomeric equilibrium in thiourea lipids impacts DNA compaction by inducing a cationic nucleation for complex assembly

Our research on lipidic vectors for transfection led us to develop thiourea lipids able to interact with DNA. Hence, we developed a series of lipopolythioureas based on the strong hydrogen bond donor ability of thiourea. More recently we have reported a branched hydroxylated bis-thiourea derivative with interesting transfecting properties. The last step of the syntheses involved a strong acidic condition, leading to an unstable product upon storage. Therefore we designed a new synthesis in mild acidic conditions. Though they exhibit the same mass, the lipids obtained in the two different conditions differ by their interaction with DNA. We therefore explored the physicochemical properties of these two lipids by different means that we describe in this article. In order to insure easier and reliable ¹³C-NMR studies of the thiourea group we have designed the synthesis of the corresponding ¹³C-labeled thiourea lipids. We have thus shown that when the lipid was submitted to mildly acidic medium; only the thiourea group was observed; while a thiourea/charged and/or uncharged iminothiol tautomeric equilibrium formed when the last step of the synthesis was submitted to low pH. NMR experiments showed that this tautomeric equilibrium could not form in polar solvents. However, UV experiments on the liposomal form of the lipopolythiourea showed the presence of the tautomers. Lipid/DNA interaction consequently differed according to the acidic treatment applied. Eventually, these results revealed that on this particular thiourea lipid, electrostatic interactions due to cationic thioureas are likely to be responsible for DNA compaction and that this tautomeric form of the thiourea could be stabilised by hydrogen bonds in a supramolecular assembly. Nevertheless, this does not reflect a general thiourea lipid/DNA interaction as other thiourea lipids that are able to compact DNA do not undergo an acidic treatment during the final stage of their synthesis.     

Binding studies of pyriproxyfen to DNA by multispectroscopic atomic force microscopy and molecular modeling methods

In this work, multispectroscopic atomic force microscopy and molecular modeling [ONIOM 2(B3LYP/6-31++G(d,p): Universal Force Field (UFF)) level] techniques were used to study the interaction between Calf-Thymus-DNA (CT-DNA) and pyriproxyfen (PYR) insecticide. The binding constant of PYR with double-strand deoxyribonucleic acid (ds-DNA) was obtained by ultraviolet-visible absorbance spectroscopy as 2.8×10(4) at 20°C. Thermodynamic parameters, that is, ΔH, ΔS°, and ΔG, were -53.82?kJ mol(-1), 96.11?J mol(-1), and -82.46?KJ mol(-1), respectively. Thermal denaturation study of DNA with PYR revealed the ΔT(m) of 3.0 and 6.0°C at r(i)=0.5 and 1.0, respectively. The Fourier transform infrared study showed a major interaction of PYR with G-C and A-T base pairs and a minor perturbation of the backbone PO(2) group. Further, PYR induces detectable changes in the circular dichroism spectrum of CT-DNA. In fluorimetric studies, the dynamic enhancement constants (k(D)) and bimolecular enhancement constant (k(B)) were calculated, which showed that the fluorescence enhancement was initiated by a static process in the ground state. The hybrid of quantum mechanical/molecular mechanics theoretical calculations revealed that the interaction is base sequence dependent, and PYR interacts more with DNA via the AT base sequence. From the data we concluded that PYR may interact with ds-DNA via two modes: intercalating and outside groove binding.     

Detection of protein–DNA interaction with a DNA probe: distinction between single-strand and double-strand DNA–protein interaction

A simple, direct method for the detection of DNA–protein interaction was developed with electrochemical methods. Single-stranded DNA (ss-DNA) probes were prepared through the chemical bonding of an oligonucleotide to a polymer film bearing carboxylic acid groups, and double-stranded DNA (ds-DNA) probes were prepared through hybridization of the complementary sequence DNA on the ss-DNA probe. Impedance spectroscopy and differential pulse voltammetry (DPV) distinguished the interaction between the DNA probes with mouse Purβ (mPurβ), an ss-DNA binding protein, and with Escherichia coli MutH, a ds-DNA binding protein. Impedance spectra obtained before and after the interaction of DNA probes with these proteins clearly showed the sequence-specific ss-DNA preference of mPurβ and the sequence-specific ds-DNA preference of MutH. The concentration dependence of proteins on the response of the DNA probes was also investigated, and the detection limits of MutH and mPurβ were 25 and 3 μg/ml, respectively. To confirm the impedance results, the variation of the current oxidation peak of adenine of the DNA probe was monitored with DPV. The formation constants of the complexes formed between the probe DNA and the proteins were estimated based on the DPV results.     

Phase Transition and Optical Properties of DNA–Gold Nanoparticle Assemblies

We review recent work on DNA-linked gold nanoparticle assemblies. The synthesis, properties, and phase behavior of such DNA–gold nanoparticle assemblies are described. These nanoparticle assemblies have strong optical extinction in the ultraviolet and visible light regions; hence, the technique is used to study the kinetics and phase transitions of DNA–gold nanoparticle assemblies. The melting transition of DNA–gold nanoparticle assemblies shows unusual trends compared to those of free DNA. The phase transitions are influenced by many parameters, such as nanoparticle size, DNA sequence, DNA grafting density, DNA linker length, interparticle distance, base pairing defects, and disorders. The physics of the DNA–gold nanoparticle assemblies can be understood in terms of the phase behavior of complex fluids, with the colloidal gold interaction potential dominated by DNA hybridization energies.     

Exploiting genomic patterns to discover new supramolecular protein assemblies

Bacterial microcompartments are supramolecular protein assemblies that function as bacterial organelles by compartmentalizing particular enzymes and metabolic intermediates. The outer shells of these microcompartments are assembled from multiple paralogous structural proteins. Because the paralogs are required to assemble together, their genes are often transcribed together from the same operon, giving rise to a distinctive genomic pattern: multiple, typically small, paralogous proteins encoded in close proximity on the bacterial chromosome. To investigate the generality of this pattern in supramolecular assemblies, we employed a comparative genomics approach to search for protein families that show the same kind of genomic pattern as that exhibited by bacterial microcompartments. The results indicate that a variety of large supramolecular assemblies fit the pattern, including bacterial gas vesicles, bacterial pili, and small heat‐shock protein complexes. The search also retrieved several widely distributed protein families of presently unknown function. The proteins from one of these families were characterized experimentally and found to show a behavior indicative of supramolecular assembly. We conclude that cotranscribed paralogs are a common feature of diverse supramolecular assemblies, and a useful genomic signature for discovering new kinds of large protein assemblies from genomic data.     

Interaction between polylysine monolayer and DNA at the air-water interface

The interaction of a polylysine amphiphile, which consists of a poly-L- or -D-lysine (1L or 1D) segment and two long alkyl chains at the C-terminus, with polynucleotides was studied with respect to the highly organized structure of polylysine assemblies on water. The results of surface pressure-area isotherm measurement showed that both of 1L and 1D formed stable monolayers on water in a neutral pH region. The secondary structure of polylysine segment for the surface monolayer was examined by means of circular dichroism and Fourier transform infrared spectroscopies. The helical structure was retained even at neutral pH, at which polylysine has been known to form a complete random coiled conformation in bulk solution. Protonated, positively charged and coiled 1L monolayer could interact electrostatically with guest polyanions including DNA in the subphase, and at the same time the conformation of the polylysine segment was converted from a random coil to an alpha-helix. Deprotonated, helical monolayers did not interact with single stranded polyadenylic acid, but with double stranded DNA. Double stranded DNA was found to interact more strongly with right-handed 1L monolayer than left-handed 1D monolayer. An obvious difference in the melting temperatures for these complexes was observed and discussed on the basis of difference in the interaction mode.     

Protein/DNA interactions involving ATF/AP1-, CCAAT-, and HiNF-D-related factors in the human H3-ST519 histone promoter: cross-competition with transcription regulatory sites in cell cycle controlled H4 and H1 histone genes.

Protein/DNA interactions of the H3-ST519 histone gene promoter were analyzed in vitro. Using several assays for sequence specificity, we established binding sites for ATF/AP1-, CCAAT-, and HiNF-D related DNA binding proteins. These binding sites correlate with two genomic protein/DNA interaction domains previously established for this gene. We show that each of these protein/DNA interactions has a counterpart in other histone genes: H3-ST519 and H4-F0108 histone genes interact with ATF- and HiNF-D related binding activities, whereas H3-ST519 and H1-FNC16 histone genes interact with the same CCAAT-box binding activity. These factors may function in regulatory coupling of the expression of different histone gene classes. We discuss these results within the context of established and putative protein/DNA interaction sites in mammalian histone genes. This model suggests that heterogeneous permutations of protein/DNA interaction elements, which involve both general and cell cycle regulated DNA binding proteins, may govern the cellular competency to express and coordinately control multiple distinct histone genes.     

DNA as a matrix of collagen fibrils

In this paper we demonstrate that DNA binds to collagen directly to form DNA–collagen complex. Our model suggests that DNA, containing well-arranged phosphate groups, helps the collagen to make ordered aggregates—fibrils. During this process hydration shell of collagen triple helix destroys and stabilizes hydration shell of ds-DNA.     

Getting a Grip on Complexes

We are witnessing tremendous advances in our understanding of the organization of life. Complete genomes are being deciphered with ever increasing speed and accuracy, thereby setting the stage for addressing the entire gene product repertoire of cells, towards understanding whole biological systems. Advances in bioinformatics and mass spectrometric techniques have revealed the multitude of interactions present in the proteome. Multiprotein complexes are emerging as a paramount cornerstone of biological activity, as many proteins appear to participate, stably or transiently, in large multisubunit assemblies. Analysis of the architecture of these assemblies and their manifold interactions is imperative for understanding their function at the molecular level. Structural genomics efforts have fostered the development of many technologies towards achieving the throughput required for studying system-wide single proteins and small interaction motifs at high resolution. The present shift in focus towards large multiprotein complexes, in particular in eukaryotes, now calls for a likewise concerted effort to develop and provide new technologies that are urgently required to produce in quality and quantity the plethora of multiprotein assemblies that form the complexome, and to routinely study their structure and function at the molecular level. Current efforts towards this objective are summarized and reviewed in this contribution.Key Words: Proteome, interactome, multiprotein assemblies, structural genomics, robotics, multigene expression, multiBac, BEVS, ACEMBL, complexomics.     

The Effect of Ca2+ Ions on DNA Compaction in the Complex with HMGB1 Nonhistone Chromosomal Protein

The analysis of absorption and circular dichroism spectra in UV and IR regions showed that Ca²⁺ ions interact with the phosphate groups of DNA and the HMGB1 protein. Not only the negatively charged C-terminal part of the protein molecule, but also its DNA-binding domains participate in the interaction with metal ions. The latter leads to a change in the mode of protein–DNA interaction. The presence of Ca²⁺ ions prevents the formation of ordered supramolecular structures specific for the HMGB1–DNA complexes but promotes intermolecular aggregation. The structure of DNA complexes with the HMGB1 protein lacking the C-terminal tail appeared to be the most sensitive to the presence of Ca²⁺ ions. These data indicate that Ca²⁺ ions play no structural role in the HMGB1–DNA complexes, and their presence is not necessary for DNA compaction in such systems.     

The effects of polyols on the thermal stability of calf thymus DNA

The effects on thermal denaturation of calf thymus DNA (ct-DNA) and its conformational changes induced by the presence in solution of different polyols, namely glycerol, i-erytritol, (−) and (+) arabitol, -mannitol, -sorbitol and myo-inositol, have been investigated by means of differential scanning calorimetry (DSC) and circular dichroism (CD). By increasing the concentration of these additives a decrease in both the denaturation enthalpy (Δ_dH) and temperature of the maximum of the denaturation peak (T_max) of DNA is observed. The values of these thermodynamic parameters depend on both the nature and concentration of the solute. The overall destabilization of DNA molecule has been related to the different capability of polyhydric alcohols to interact with the polynucleotide solvation sites replacing water and to the modification of the electrostatic interactions between the polynucleotide and its surrounding atmosphere of counterions. The particular behaviour of (−) arabitol, which showed a much greater destabilizing ability compared to the other polyols, was further investigated and attributed to a direct more effective interaction with the double helix of DNA. CD spectra showed only a slight alteration of DNA-B structure in the presence of all the molecules here studied, except for (−) arabitol where the DNA molecule seems to undergo a meaningful conformational change. The salt concentration dependence of DNA thermal stability in the presence of (−) arabitol indicates a conformational change of polynucleotide towards a more extended conformation.