Interactions of chromomycin A3 and mithramycin with the sequence d(TAGCTAGCTA)2

Anti-cancer antibiotics, chromomycin A3 (CHR) and mithramycin (MTR) inhibit DNA directed RNA synthesis in vivo by binding reversibly to template DNA in the minor groove with GC base specificity, in the presence of divalent cations like Mg2+. Under physiological conditions, (drug)2Mg2+ complexes formed by the antibiotics are the potential DNA binding ligands. Structures of CHR and MTR differ in their saccharide residues. Scrutiny of the DNA binding properties reveal significant differences in their sequence selectivity, orientation and stoichiometry of binding. Here, we have analyzed binding and thermodynamic parameters for the interaction of the antibiotics with a model oligonucleotide sequence, d(TAGCTAGCTA)2 to understand the role of sugars. The oligomer contains two potential binding sites (GpC) for the ligands. The study illustrates that the drugs bind differently to the sequence. (MTR)2Mg2+ binds to both sites whereas (CHR)2Mg2+ binds to a single site. UV melting profiles for the decanucleotide saturated with the ligands show that MTR bound oligomer is highly stabilized and melts symmetrically. In contrast, with CHR, loss of symmetry in the oligomer following its association with a single (CHR)2Mg2+ complex molecule leads to a biphasic melting curve. Results have been interpreted in the light of saccharide dependent differences in ligand flexibility between the two antibiotics.     

Interaction of mithramycin with chromatin

Mithramycin (MTR) is an anti-cancer antibiotic that blocks the macromolecular biosynthesis via reversible interaction with DNA template in the presence of bivalent metal ion such as Mg2+. In absence of DNA, mithramycin forms two types of complexes with Mg2+, complex I (with 1:1 stoichiometry in terms of MTR: Mg2+) and complex II (with 1:2 stoichiometry in terms of MTR: Mg2+). In an eukaryotic system, the drug would interact with chromatin, a protein-DNA complex. We have employed the spectroscopic techniques such as absorption and fluorescence to study the interaction of MTR: Mg2+ complexes with rat liver chromatin. In this report, we have shown that the two types of ligands have different binding potentials with the same chromatin. This supports our proposition that complexes I and II, are different molecular species. We have also shown that the histone protein(s) reduce the binding potential and the number of available sites for both ligands.     

Association of antitumor antibiotic Mithramycin with Mn2+ and the potential cellular targets of Mithramycin after association with Mn2+

Mithramycin (MTR), an aureolic acid group of antitumor antibiotic is used for the treatment of several types of tumors. We have reported here the association of MTR with an essential micronutrient, manganese (Mn²⁺). Spectroscopic methods have been used to characterize and understand the kinetics and mechanism of complex formation between them. MTR forms a single type of complex with Mn²⁺ in the mole ratio of 2:1 [MTR: Mn²⁺] via a two step kinetic process. Circular dichroism (CD) spectroscopic study indicates that the complex [(MTR)₂ Mn²⁺] has a right-handed twist conformation similar in structure with the complexes reported for Mg²⁺ and Zn²⁺. This conformation allows binding via minor groove of DNA with (G, C) base preference during the interaction with double-stranded B-DNA. Using absorbance, fluorescence, and CD spectroscopy we have shown that [(MTR)₂ Mn²⁺] complex binds to double-stranded DNA with an apparent dissociation constant of 32?μM and binding site size of 0.2 (drug/nucleotide). It binds to chicken liver chromatin with apparent dissociation constant value 298?μM. Presence of histone proteins in chromatin inhibits the accessibility of the complex for chromosomal DNA. We have also shown that MTR binds to Mn²⁺ containing metalloenzyme manganese superoxide dismutase from Escherichia coli.     

Role of magnesium ion in the interaction between chromomycin A3 and DNA: binding of chromomycin A3-Mg2+ complexes with DNA.

Chromomycin A3 is an antitumor antibiotic which blocks macromolecular synthesis via reversible interaction with DNA template only in the presence of divalent metal ions such as Mg2+. The role of Mg2+ in this antibiotic-DNA interaction is not well understood. We approached the problem in two steps via studies on the interaction of (i) chromomycin A3 and Mg2+ and (ii) chromomycin A3-Mg2+ complex(es) and DNA. Spectroscopic techniques such as absorption, fluorescence, and CD were employed for this purpose. The results could be summed up in two parts. Absorption, fluorescence, and CD spectra of the antibiotic change upon addition of Mg2+ due to complex formation between them. Analysis of the quantitative dependence of change in absorbance of chromomycin A3 (at 440 nm) upon input concentration of Mg2+ indicates formation of two types of complexes with different stoichiometries and formation constants. Trends in change of fluorescence and CD spectroscopic features of the antibiotic in the presence of Mg2+ at different concentrations further corroborate this result. The two complexes are referred to as complex I (with 1:1 stoichiometry in terms of chromomycin A3:Mg2+) and complex II (with 2:1 stoichiometry in terms of chromomycin A3:Mg2+), respectively, in future discussions. The interactions of these complexes with calf thymus DNA were examined to check whether they bind differently to the same DNA. Evaluation of binding parameters, intrinsic binding constants, and binding stoichiometry, by means of spectrophotometric and fluorescence titrations, shows that they are different. Distinctive spectroscopic features of complexes I and II, when they are bound to DNA, also support that they bind differently to the above DNA. Measurement of thermodynamic parameters characterizing their interactions with calf thymus DNA shows that complex I-DNA interaction is exothermic, in contrast to complex II-DNA interaction, which is endothermic. This feature implies a difference in the molecular nature of the interactions between the complexes and calf thymus DNA. These observations are novel and significant to understand the antitumor property of the antibiotic. They are also discussed to provide explanations for the earlier reports that in some cases appeared to be contradictory.     

Laser Raman spectra of calf thymus chromatin and its constituents.

Extensive Raman measurements have been made on calf thymus chromatin, core chromatin, the (H3,H4)/DNA complex, and isolated DNA. The results indicate that the alpha-helical content of the nucleosomal histones gradually increases as they form the heterocomplexes that lead to the formation of the octameric nucleosome core. The secondary structure of the latter is not modified as it binds to DNA. The spectra indicate that the DNA essentially retains its B conformation in nucleosomes, although slight changes probably occur in the ribose-phosphate backbone. No specific interactions between the nucleosomal histones and DNA can be established from the spectra, but histone H1 possibly interacts selectively with the thymine bases.     

On the structure and dynamics of the complex of the nucleosome and the linker histone

Several different models of the linker histone (LH)–nucleosome complex have been proposed, but none of them has unambiguously revealed the position and binding sites of the LH on the nucleosome. Using Brownian dynamics-based docking together with normal mode analysis of the nucleosome to account for the flexibility of two flanking 10 bp long linker DNAs (L-DNA), we identified binding modes of the H5-LH globular domain (GH5) to the nucleosome. For a wide range of nucleosomal conformations with the L-DNA ends less than 65 Å apart, one dominant binding mode was identified for GH5 and found to be consistent with fluorescence recovery after photobleaching (FRAP) experiments. GH5 binds asymmetrically with respect to the nucleosomal dyad axis, fitting between the nucleosomal DNA and one of the L-DNAs. For greater distances between L-DNA ends, docking of GH5 to the L-DNA that is more restrained and less open becomes favored. These results suggest a selection mechanism by which GH5 preferentially binds one of the L-DNAs and thereby affects DNA dynamics and accessibility and contributes to formation of a particular chromatin fiber structure. The two binding modes identified would, respectively, favor a tight zigzag chromatin structure or a loose solenoid chromatin fiber.     

Acidic pentapeptide phosphorylated in vitro by calf thymus protein kinase NII binds to DNA in the presence of Mg2+ cations

The pentapeptide pyroGlu-Ala-Glu-Ser-Asn has been synthetized and phosphorylated in vitro at level of serine by protein kinase NII isolated from calf thymus chromatin. It is noteworthy that the calf thymus kinase NII shows a remarkable affinity for this peptide. The [32P]peptide is able to bind to several DNAs in the presence of Mg2+ (lambda phage, calf thymus, pBR540 plasmid). This binding appears not specific with regard to the type of DNA and its base sequence. These data support the hypothesis that phosphorylated acidic domains of nuclear nonhistone proteins could bind directly to DNA in the presence of Mg2+ cations.     

Interaction of mithramycin with DNA. Evidence that mithramycin binds to DNA as a dimer in a right-handed screw conformation.

The CD and fluorescence properties of mithramycin have been used to follow its complexation to cations such as Mg2+ and Zn2+ and the binding of these complexes to DNA. At low concentration (less than 2 microM) in aqueous solution, mithramycin is always in the dimeric state, the conformation of the dimer being either a right-handed screw when the dimer is neutral, or a left-handed screw when the dimer is negatively charged. In the deprotonated state the dimer can bind one cation forming a complex [M2+(Mit-)2] which has a right-handed screw conformation. The stability constants of the complex at 37 degrees C in 0.1 M KCl are 4 x 10(5) and 1 x 10(6) for Mg2+ and Zn2+, respectively. The complex in the right-handed screw conformation binds DNA. In this case the stability constants of the complex [M2+ (Mit-)2] increase and are 3.6 x 10(6) and 1.2 x 10(7) for Mg2+ and Zn2+, respectively.     

Differential interactions of antitumor antibiotics chromomycin A(3) and mithramycin with d(TATGCATA)(2) in presence of Mg(2+)

The antitumor antibiotics chromomycin A(3) (CHR) and mithramycin (MTR) are known to inhibit macromolecular biosynthesis by reversibly binding to double stranded DNA with a GC base specificity via the minor groove in the presence of a divalent cation such as Mg(2+). Earlier reports from our laboratory showed that the antibiotics form two types of complexes with Mg(2+): complex I with 1:1 stoichiometry and complex II with 2:1 stoichiometry in terms of the antibiotic and Mg(2+). The binding potential of an octanucleotide, d(TATGCATA)(2), which contains one potential site of association with the above complexes of the two antibiotics, was examined using spectroscopic techniques such as absorption, fluorescence, and circular dichroism. We also evaluated thermodynamic parameters for the interaction. In spite of the presence of two structural moieties of the antibiotic in complex II, a major characteristic feature was the association of a single ligand molecule per molecule of octameric duplex in all cases. This indicated that the modes of association for the two types of complexes with the oligomeric DNA were different. The association was dependent on the nature of the antibiotics. Spectroscopic characterization along with analysis of binding and thermodynamic parameters showed that differences in the mode of recognition by complexes I and II of the antibiotics with polymeric DNA existed at the oligomeric level. Analysis of the thermodynamic parameters led us to propose a partial accommodation of the ligand in the groove without the displacement of bound water molecules and supported earlier results on the DNA structural transition from B --> A type geometry as an obligatory requirement for the accommodation of the bulkier complex II of the two drugs. The role of the carbohydrate moieties of the antibiotics in the DNA recognition process was indicated when we compared the DNA binding properties with the same type of Mg(2+) complex for the two antibiotics.     

Intercalation of a Zn(II) complex containing ciprofloxacin drug between DNA base pairs

In this study, an attempt has been made to study the interaction of a Zn(II) complex containing an antibiotic drug, ciprofloxacin, with calf thymus DNA using spectroscopic methods. It was found that Zn(II) complex could bind with DNA via intercalation mode as evidenced by: hyperchromism in UV–Vis spectrum; these spectral characteristics suggest that the Zn(II) complex interacts with DNA most likely through a mode that involves a stacking interaction between the aromatic chromophore and the base pairs of DNA. DNA binding constant (K_b = 1.4 × 10⁴ M^?1) from spectrophotometric studies of the interaction of Zn(II) complex with DNA is comparable to those of some DNA intercalative polypyridyl Ru(II) complexes 1.0 ?4.8 × 10⁴ M^?1. CD study showed stabilization of the right-handed B form of DNA in the presence of Zn(II) complex as observed for the classical intercalator methylene blue. Thermodynamic parameters (ΔH < 0 and ΔS < 0) indicated that hydrogen bond and Van der Waals play main roles in this binding prose. Competitive fluorimetric studies with methylene blue (MB) dye have shown that Zn(II) complex exhibits the ability of this complex to displace with DNA-MB, indicating that it binds to DNA in strong competition with MB for the intercalation.