Depicting the binding of furazolidone/furacilin with DNA by multiple spectroscopies,voltammetric as well as molecular docking

The interaction between DNA and furazolidone/furacillin was investigated using various analytical techniques including spectroscopy and electroanalysis and molecular modelling. With the aid of acridine orange (AO), the fluorescence lifetimes of DNA–AO, DNA–furazolidone/furacillin–AO remained almost the same, which proved that the ground state complex was formed due to furazolidone/furacillin binding with DNA. Circular dichroism spectra and Fourier transform infrared spectroscopy showed that the second structure of DNA changed. Viscosity experiments presented that relative viscosity of DNA was increased with the increasing concentrations of furazolidone and almost unchanged for furacilin. In addition, the results of melting temperature (T_m), ionic strength, site competition experiments, cyclic voltammetry, and molecular docking all proved the intercalation binding mode for furazolidone and groove binding mode for furacilin. The binding constants (K_a) obtained from Wolfe–Shimmer equation were calculated as 3.66 × 10⁴ L mol^?1 and 3.95 × 10⁴ L mol^?1 for furazolidone–DNA and furacilin–DNA, respectively.     

Identification of a reactive intermediate of furazolidone formed by swine liver microsomes

Furazolidone (N-(5-nitro-2-furfurylidene)-3-amino-2-oxazolidone) is metabolized by swine liver microsomes under aerobic and anaerobic conditions (rate: 2.55 and 3.25 nmol/mg protein/min, respectively). Covalent binding to microsomal protein amounted aerobically to 0.29 nmol/mg protein/min. Of all amino acids tested, only addition of cysteine to the incubation mixture decreased microsomal protein binding of furazolidone, indicating that covalent binding may occur at protein thiol groups. Two known metabolites of furazolidone, 3-(4-cyano-2-oxobutylidene-amino)-2-oxazolidone and 2,3 dihydro-3-cyano-methyl-2-hydroxyl-5-nitro-1 alpha,2-di(2-oxo-oxazolidin-3-yl) iminomethyl-furo[2,3-b] furan, were minor metabolites. At least 50% of total metabolites is formed by swine liver microsomes via a reductive process of furazolidone as indicated by the formation of a furazolidone-mercaptoethanol conjugate after the addition of mercaptoethanol to the incubation mixture. The conjugate was identified as 3-(4-cyano-3-beta-hydroxyethylmercapto-2-oxobutylidene amino)-2-oxazolidone, indicating that the open-chain acrylonitrile-derivative is the reactive intermediate of furazolidone which also may be responsible for interaction with protein.     

Radiomimetic property of furazolidone and the caffeine enhancement of its lethal action on the vibrios

Sensitivities of the strains belonging to four vibrio biotypes to the action of furazolidone were investigated. Vibrio cholerae (classical) was most and Vibrio parahaemolyticus least sensitive to this drug. Statistical analyses revealed significant differences between any two of the four types of vibrio in respect of their sensitivity to furazolidone. The drug was radiomimetic in action, the doses of UV light (D_UV) and furazolidone (D_f) required for 10% survival of the vibrios being correlated by the equation, D_f = 0.28 exp. (0.008 D_UV). Caffeine exhibited lethal synergism with furazolidone and the synergistic effect depended on the mode of caffeine treatment, the effect being maximum when caffeine was present along with and also after furazolidone treatment. UV spectrophotometric study revealed that caffeine did not bind with native DNA but did so with denatured DNA resulting in a bathochromic shift and a quenching of the caffeine absorption maximum at 209.4 nm. The binding isotherm (Scatchard plot) indicated the presence of a heterogeneity in the binding sites and that the parameters for the strongest mode of binding were n = 0.254 and k = 7.5 × 10⁵ M^?1.     

Structural reorganization and the cooperative binding of single-stranded telomere DNA in Sterkiella nova

In Sterkiella nova, alpha and beta telomere proteins bind cooperatively with single-stranded DNA to form a ternary alpha.beta.DNA complex. Association of telomere protein subunits is DNA-dependent, and alpha-beta association enhances DNA affinity. To further understand the molecular basis for binding cooperativity, we characterized several possible stepwise assembly pathways using isothermal titration calorimetry. In one path, alpha and DNA first form a stable alpha.DNA complex followed by the addition of beta in a second step. Binding energy accumulates with nearly equal free energy of association for each of these steps. Heat capacity is nonetheless dramatically different, with DeltaCp = -305 +/- 3 cal mol(-1) K(-1) for alpha binding with DNA and DeltaCp = -2010 +/- 20 cal mol(-1) K(-1) for the addition of beta to complete the alpha.beta.DNA complex. By examining alternate routes including titration of single-stranded DNA with a preformed alpha.beta complex, a significant portion of binding energy and heat capacity could be assigned to structural reorganization involving protein-protein interactions and repositioning of the DNA. Structural reorganization probably affords a mechanism to regulate high affinity binding of telomere single-stranded DNA with important implications for telomere biology. Regulation of telomere complex dissociation is thought to involve post-translational modifications in the lysine-rich C-terminal portion of beta. We observed no difference in binding energetics or crystal structure when comparing complexes prepared with full-length beta or a C-terminally truncated form, supporting interesting parallels between the intrinsically disordered regions of histones and this portion of beta.     

Nonspecific binding of lac repressor to DNA. I. An absorption and circular dichroism study

Nonspecific binding of lac repressor on DNA has been studied by absorption and circular dichroism (CD) spectroscopies. In a first step, the complex formation is accompanied by an absorption difference spectrum and a change of the CD signal of the DNA. The absorption difference spectrum is mainly due to a spectral change of the DNA. The variation of the CD signal has been analyzed according to a model calculation, which takes into account the fact that the excluded site is shorter than the perturbed site. We found that in this first step one repressor can bind every 14 +/- 2 base-pairs, whereas one repressor perturbs 22 +/- 2 base-pairs. In a second step, more repressor can bind on DNA, but without further change in the absorption and CD spectrum, indicating that another binding process occurs. The model calculation developed here is general for all binding processes inducing a perturbation over a length of DNA longer than that of the excluded site.     

Noncovalent DNA binding of bis(1,10-phenanthroline)copper(I) and related compounds

The noncovalent DNA binding of the bis(1,10-phenanthroline)copper(I) complex [(Phen)2CuI] was examined under anaerobic conditions by absorption and circular dichroism spectroscopy, and viscometry, as a function of phenanthroline concentration. Analyses according to the McGhee-von Hippel method indicated that binding exhibited both neighbor-exclusion and positive cooperativity effects, with a neighbor-exclusion parameter n approximately 2 and a cooperativity parameter omega approximately 4. The association constant for (Phen)2CuI binding decreased with increasing concentration of phenanthroline in excess over that required to stoichiometrically generate (Phen)2CuI, indicating that free phenanthroline was a weak competitive inhibitor of (Phen)2CuI binding. The maximal association constant for DNA binding of (Phen)2CuI in 0.2 M NaCl and 9.8% ethanol, extrapolated to zero concentration of excess phenanthroline, was 4.7 x 10(4) M-1 (DNA base pairs). The magnitude of the neighbor-exclusion parameter, the changes in spectral properties of (Phen)2CuI induced by DNA binding, and the increase in DNA solution viscosity upon (Phen)2CuI addition are consistent with a model for DNA binding by (Phen)2CuI involving partial intercalation of one phenanthroline ring of the complex between DNA base pairs in the minor groove as suggested previously [Veal & Rill (1989) Biochemistry 28, 3243-3250]. Viscosity measurements indicated that the mono(phenanthroline)copper(I) complex also binds to DNA by intercalation; however, no spectroscopic or viscometric evidence was found for DNA binding of free phenanthroline or the bis(2,9-dimethyl-1,10-phenanthroline)copper(I) complex. DNA binding of free phenanthroline may be cooperative and induced by prior binding of (Phen)2CuI.     

Stable binding of recA protein to duplex DNA. Unraveling a paradox

recA protein binding to duplex DNA is a complicated, multistep process. The final product of this process is a stably bound complex of recA protein and extensively unwound double-stranded DNA. recA monomers within the complex hydrolyze ATP with an apparent kcat of approximately 19-22 min-1. Once the final binding state is achieved, binding and ATP hydrolysis by this complex becomes pH independent. The weak binding of recA protein to duplex DNA reported in previous studies does not, therefore, reflect an intrinsically unfavorable binding equilibrium. Instead, this apparent weak binding reflects a slow step in the association pathway. The rate-limiting step in this process involves the initiation rather than the propagation of DNA binding and unwinding. This step exhibits no dependence on recA protein concentration at pH 7.5. Extension or propagation of the recA filament is fast relative to the overall process. Initiation of binding is pH dependent and represents a prominent kinetic barrier at pH 7.5. ATP hydrolysis occurs only after the duplex DNA is unwound. The binding density of recA protein on double-stranded DNA is approximately one monomer/4 base pairs. A model for this process is presented. These results provide an explanation for several paradoxical observations about recA protein-promoted DNA strand exchange. In particular, they demonstrate that there is no thermodynamic requirement for dissociation of recA protein from the heteroduplex DNA product of strand exchange.     

Function and disruption of DNA methyltransferase 3a cooperative DNA binding and nucleoprotein filament formation

The catalytic domain of Dnmt3a cooperatively multimerizes on DNA forming nucleoprotein filaments. Based on modeling, we identified the interface of Dnmt3a complexes binding next to each other on the DNA and disrupted it by charge reversal of critical residues. This prevented cooperative DNA binding and multimerization of Dnmt3a on the DNA, as shown by the loss of cooperative complex formation in electrophoretic mobility shift assay, the loss of cooperativity in DNA binding in solution, the loss of a characteristic 8- to 10-bp periodicity in DNA methylation and direct imaging of protein-DNA complexes by scanning force microscopy. Non-cooperative Dnmt3a-C variants bound DNA well and retained methylation activity, indicating that cooperative DNA binding and multimerization of Dnmt3a on the DNA are not required for activity. However, one non-cooperative variant showed reduced heterochromatic localization in mammalian cells. We propose two roles of Dnmt3a cooperative DNA binding in the cell: (i) either nucleofilament formation could be required for periodic DNA methylation or (ii) favorable interactions between Dnmt3a complexes may be needed for the tight packing of Dnmt3a at heterochromatic regions. The complex interface optimized for tight packing would then promote the cooperative binding of Dnmt3a to naked DNA in vitro.     

Site selective bis-intercalation of a homodimeric thiazole orange dye in DNA oligonucleotides.

We have used one and two dimensional 1H NMR spectroscopy to characterize the binding of a homodimeric thiazole orange dye, 1,1'-(4,4,8,8-tetramethyl-4,8-diaza-undecamethylene)-bis-4- (3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)-quinolin ium tetraiodide (TOTO), to various double stranded DNA oligonucleotides. TOTO binds strongly to all the oligonucleotides used, but usually more than one complex is observed and exchange between different binding sites broadens the lines in the NMR spectra. Complete precipitation occurs when TOTO is bound to small oligonucleotides. Binding to larger oligonucleotides occurs by bis-intercalation. The 1:1 complex of TOTO with the oligonucleotide d(CCGACTGATGC):d (GCATCAGTCGG) gave only one complex that was shown to be a bis-intercalation in the CTGA:TCAG binding site. The binding to this site was also characterized by studying the TOTO complex with the d(CCGCTGAGC):d(GCTCAGCGG) oligonucleotide. NOE connectivities and molecular modelling were used to characterize the complex. The 1:1 complex of TOTO with the oligonucleotide d(CCGCTAGCG):d(CGCTAGCGG) containing a CTAG:CTAG binding site was similarly characterized by NMR. It was concluded that the binding of TOTO to larger oligonucleotides is site selective with CTAG:CTAG as the preferred binding site.     

DNA conformational effects on the interaction of netropsin with A-tract sequences

The influence of cosolutes and DNA sequence on the interaction of netropsin with three duplexes has been studied by isothermal titration calorimetry. In buffer, netropsin forms two complexes with a net stoichiometry of 1:1 in the minor groove of the oligonucleotide (GCGCGAATTCGCGC)2. One complex has a weaker affinity and is more enthalpically favored relative to the other one, consistent with previous studies [Freyer, M. W., et al. (2006) Biophys. Chem. 126, 186-196]. With the cosolutes betaine and 2-methyl-2,4-pentanediol, the enthalpy and heat capacity changes indicate that the complex with weaker affinity is disfavored relative to the complex with higher affinity. With (CGCGCAATTGCGCG)2, netropsin has one binding mode in buffer, and complex formation is not influenced by the cosolutes. The similarities of the enthalpy and heat capacity changes suggest that netropsin interacts similarly with these two oligonucleotides in the presence of cosolutes. The oligonucleotide (GCGCAAATTTGCGC)2 also forms two complexes with netropsin, and the complex with weaker affinity is again disfavored by the cosolutes. Thus, the interaction of netropsin with these A/T binding sites is influenced both by the bases adjacent to the binding site and by cosolutes. We suggest that these two factors influence the conformation of the minor-groove binding site of DNA.     

Oxidative DNA cleavage by Schiff base tetraazamacrocyclic oxamido nickel(II) complexes

Nickel is considered a weak carcinogen. Some researches have shown that bound proteins or synthetic ligands may increase the toxic effect of nickel ions. A systematic study of ligand effects on the interaction between nickel complexes and DNA is necessary. Here, we compared the interactions between DNA and six closely related Schiff base tetraazamacrocyclic oxamido nickel(II) complexes NiL(1-3a,1-3b). The structure of one of the six complexes, NiL(3b) has been characterized by single crystal X-ray analysis. All of the complexes can cleave plasmid DNA under physiological conditions in the presence of H(2)O(2). NiL(3b) shows the highest DNA cleavage activity. It can convert supercoiled DNA to nicked DNA then linear DNA in a sequential manner as the complex concentration or reaction time is increased. The cleavage reaction is a typical pseudo-first-order consecutive reaction with the rate constants of 3.27+/-0.14h(-1) (k(1)) and 0.0966+/-0.0042h(-1) (k(2)), respectively, when a complex concentration of 0.6mM is used. The cleavage mechanism between the complex and plasmid DNA is likely to involve hydroxyl radicals as reactive oxygen species. Circular dichronism (CD), fluorescence spectroscopy and gel electrophoresis indicate that the complexes bind to DNA by partial intercalative and groove binding modes, but these binding interactions are not the dominant factor in determining the DNA cleavage abilities of the complexes.     

Spectroscopy, cytotoxicity and DNA-binding of the lanthanum(III) complex of an L-valine derivative of 1,10-phenanthroline

The interaction of the lanthanum(III) La(III)-L (L=N,N'-bis-(1-carboxy-2-methylpropyl)-1,10-phenanthroline-2,9-dimethanamine) complex with calf thymus DNA was studied by electronic spectra, fluorescence spectra and circular dichroic spectra. The La(III)-L complex was assayed for antitumor activity in vitro against the HL-60 (the human leucocytoma) cells, HCT-8 (the human coloadenocarcinoma) cells, BGC-823 (the human carcinoma of stomach) cells, Bel-7402 (the human liver carcinoma) cells and KB (the human nasopharyngeal carcinoma) cells. The results show that the La(III)-L complex has activity against HL-60 cells, Bel-7402 cells and KB cells. Moreover, it is slightly more effective against Bel-7402 cell line than cisplatin. Using ethidium bromide as a fluorescence probe, the binding mode of the La(III)-L complex to calf-thymus DNA was studied spectroscopically. For comparison, the same measurements were carried out with La(III)-Phen [La(III)-1,10-phenanthroline complex] and La(III)-Val [La(III)-L-valine complex]. The results indicate that the La(III)-L and La(III)-Phen complexes possibly interact with calf-thymus DNA by both intercalative and coordination binding, whereas the La(III)-Val complex interacts with calf-thymus DNA by coordination binding. Kinetics of binding of the three complexes to DNA is for the first time studied using ethidium bromide as a fluorescence probe with stopped-flow spectrophotometer under pseudo-first-order condition. The strong two-step mechanisms in the process of the La(III)-L and La(III)-Phen complexes and one step in the process of the complex La(III)-Val interacting with DNA are observed, and the k(obs) (observed pseudo-first-order rate constant) and E(a) (observed energy of activation) values of binding to DNA are obtained.     

The cyclic AMP (cAMP)-cAMP receptor protein complex functions both as an activator and as a corepressor at the tsx-p2 promoter of Escherichia coli K-12.

The tsx-p2 promoter is one of at least seven Escherichia coli promoters that are activated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex and negatively regulated by the CytR repressor. DNase I footprinting assays were used to study the interactions of these regulatory proteins with the tsx-p2 promoter region and to characterize tsx-p2 regulatory mutants exhibiting an altered response to CytR. We show that the cAMP-CRP activator complex recognizes two sites in tsx-p2 that are separated by 33 bp: a high-affinity site (CRP-1) overlaps the -35 region, and a low-affinity site (CRP-2) is centered around position -74 bp. The CytR repressor protects a DNA segment that is located between the two CRP sites and partially overlaps the CRP-1 target. In combination, the cAMP-CRP and CytR proteins bind cooperatively to tsx-p2, and the nucleoprotein complex formed covers a region of 78 bp extending from the CRP-2 site close to the -10 region. The inducer for the CytR repressor, cytidine, does not prevent in vitro DNA binding of CytR, but releases the repressor from the nucleoprotein complex and leaves the cAMP-CRP activator bound to its two DNA targets. Thus, cytidine interferes with the cooperative DNA binding of cAMP-CRP and CytR to tsx-p2. We characterized four tsx-p2 mutants exhibiting a reduced response to CytR; three carried mutations in the CRP-2 site, and one carried a mutation in the region between CRP-1 and the -10 sequence. Formation of the cAMP-CRP-CytR DNA nucleoprotein complex in vitro was perturbed in each mutant. These data indicate that the CytR repressor relies on the presence of the cAMP-CRP activator complex to regulate tsx-p2 promoter activity and that the formation of an active repression complex requires the combined interactions of cAMP-CRP and CytR at tsx-p2.     

Nucleosomal structure of undamaged DNA regions suppresses the non-specific DNA binding of the XPC complex

The XPC protein complex is a DNA damage detector of human nucleotide excision repair (NER). Although the XPC complex specifically binds to certain damaged sites, it also binds to undamaged DNA in a non-specific manner. The addition of a large excess of undamaged naked DNA competitively inhibited the specific binding of the XPC complex to (6-4) photoproducts and the NER dual incision step in cell-free extracts. In contrast, the addition of undamaged nucleosomal DNA as a competitor suppressed both of these inhibitory effects. Although nucleosomes positioned on the damaged site inhibited the binding of the XPC complex, the presence of nucleosomes in undamaged DNA regions may help specific binding of the XPC complex to damaged sites by excluding its non-specific binding to undamaged DNA regions.     

Single-molecule investigation of the T4 bacteriophage DNA polymerase holoenzyme: multiple pathways of holoenzyme formation

In T4 bacteriophage, the DNA polymerase holoenzyme is responsible for accurate and processive DNA synthesis. The holoenzyme consists of DNA polymerase gp43 and clamp protein gp45. To form a productive holoenzyme complex, clamp loader protein gp44/62 is required for the loading of gp45, along with MgATP, and also for the subsequent binding of polymerase to the loaded clamp. Recently published evidence suggests that holoenzyme assembly in the T4 replisome may take place via more than one pathway [Zhuang, Z., Berdis, A. J., and Benkovic, S. J. (2006) Biochemistry 45, 7976-7989]. To demonstrate unequivocally whether there are multiple pathways leading to the formation of a productive holoenzyme, single-molecule fluorescence microscopy has been used to study the potential clamp loading and holoenzyme assembly pathways on a single-molecule DNA substrate. The results obtained reveal four pathways that foster the formation of a functional holoenzyme on DNA: (1) clamp loader-clamp complex binding to DNA followed by polymerase, (2) clamp loader binding to DNA followed by clamp and then polymerase, (3) clamp binding to DNA followed by clamp loader and then polymerase, and (4) polymerase binding to DNA followed by the clamp loader-clamp complex. In all cases, MgATP is required. The possible physiological significance of the various assembly pathways is discussed in the context of replication initiation and lagging strand synthesis during various stages of T4 phage replication.