Interaction of anthracyclines with DNA and chromosomes

Daunomycin and adriamycin were previously found to produce Q-like banding patterns on chromosomes. The interaction of several anthracyclines with both natural and synthetic DNAs and chromosomes has been investigated in more detail. Daunomycin fluorescence is almost completely quenched by natural DNAs with varying base composition from 31 to 72% G-C and by the alternating polymer poly-d(G-C)·poly-d(G-C). In contrast, daunomycin fluorescence is quenched by only 50% when the dye interacts with synthetic A-T polymers. Thus, differential quenching of daunomycin fluorescence can account for the production of bright bands at contiguous A-T sequences along the chromosome. Slight differences in fluorescence quenching between the repeating and homopolymeric A-T duplex DNAs were observed which can be attributed to differences in affinity of daunomycin for these DNAs. The aminosugar moiety of daunomycin, daunosamine, increases the binding of daunomycin to DNA and also enhances chromosome banding. — Nogalamycin, which displays no differential quenching with the different DNAs in solution, also fails to produce bands on chromosomes. — These findings suggest that non-random nucleotide sequence arrangements along the chromosome are a basic determinant for dye interaction to produce the observed banding patterns. Specific banding procedures may determine the accessibility of these sites within the chromosomal DNA.     

Molecular dynamics studies of trinucleotide repeat DNA involved in neurodegenerative disorders.

Expansion of trinucleotide repeat DNA of the classes CAG-CTG, CGG-CCG and GAA-TTC are found to be associated with several neurodegenerative disorders. Different mechanisms have been attributed to the expansion of triplets, mainly involving the formation of alternate secondary structures by such repeats. This paper reports the molecular dynamics simulation of triplet repeat DNA sequences to study the basic structural features of DNA that are responsible for the formation of structures such as hairpins and slip-strand DNA leading to expansion. All the triplet repeat sequences studied were found to be more flexible compared to the control sequence unassociated with disease. Moreover, flexibility was found to be in the order CAG-CTG > CGG-CCG approximately GAA-TTC, the highly flexible CAG-CTG repeat being the most common cause of neurodegenerative disorders. In another simulation, a single G-C to T-A mutation at the 9th position of the CAG-CTG repeat exhibited a reduction in bending compared to the pure 15-mer CAG-CTG repeat. EPM1 dodecamer repeat associated with the pathogenesis of progressive myoclonus epilepsy was also simulated and showed flexible nature suggesting a similar expansion mechanism.     

Identification of structural domains in protein C involved in its interaction with thrombin-thrombomodulin on the surface of endothelial cells.

The structural domains of protein C involved in its interaction with thrombin-thrombomodulin on the endothelial cell surface have been investigated using isolated intact domains of bovine protein C produced from controlled proteolytic digests of the protein. The fragments investigated include the gamma-carboxyglutamic acid (Gla)-rich module, the two epidermal growth factor (EGF)-like modules, and a fragment consisting of the Gla and the two EGF-like modules. The effects of these fragments on the catalytic efficiency (Km and Vmax) of activation of protein C by the endothelial cell surface thrombin-thrombomodulin complex (IIa-TM) have been evaluated in vitro using a stirred microcarrier cell culture of bovine aortic endothelial cells and purified proteins. Neither the Gla nor the two EGF-like modules alone had any discernible effect on protein C activation. The intact Gla-EGF fragment, however, inhibited protein C activation. The results are consistent with a rapid equilibrium competitive inhibition model, in which the Gla-EGF fragment competes with protein C for binding to IIa-TM, and indicate that the Gla-EGF fragment alone accounts for most of the binding energy of intact protein C for IIa-TM. In addition, a requirement for the Gla residues of protein C for binding is implied by the observation that heat-decarboxylated Gla-EGF fragment was not an inhibitor of protein C activation. In addition, chloromethyl ketone-inactivated activated protein C was found to bind to IIa-TM with the same affinity as protein C, suggesting that the changes which occur in protein C upon activation do not affect that part of the protein responsible for binding to IIa-TM, that is the Gla-EGF region. The Gla-EGF region from factor X also weakly inhibited the IIa-TM activation of protein C.     

DNA unwinding and inhibition of T4 DNA ligase by anthracyclines.

The ability to alter DNA tertiary structure of ten anthracycline derivatives whose antitumor potency is known was studied by an assay that makes use of nicked circular DNA and bacteriophage T4 DNA ligase. This assay allows the detection of tertiary structure alterations caused by DNA binding of both intercalating and non-intercalating drugs. The determination of these events can be obtained at different temperatures in the range of activity of DNA ligase. The results indicate that anthracyclines alter the DNA tertiary structure but this property does not correlate with their cytotoxic or antitumor activities. An additional interesting finding was that several anthracyclines inhibit T4 DNA ligase. The inhibition can be complete and is a cubic function of drug concentration. The inhibition of DNA ligase does not correlate with the ability of anthracyclines to alter the tertiary structure of DNA but is dependent from the presence of an amino group on the sugar ring.     

Molecular interaction analysis of cigarette smoke carcinogens NNK and NNAL with enzymes involved in DNA repair pathways: An in silico approach

DNA damage occurs almost all the times in cells, but is repaired also continuously. Occurrence of all these mutations and their accumulation in one cell which finally becomes tumorigenic/carcinogenic appears possible if the DNA repair mechanism is hampered. We hypothesize that alterations in DNA repair pathways, either all or at least at one i.e. genetic, translational or posttranslational level, becomes quite imperative for the initiation and progression of Cancer. Therefore, we investigated the interaction capability of some carcinogens with the enzymes involved in the DNA repair mechanisms. Cigarette smoke''s derivatives like NNK and NNAL are well established carcinogens. Hence, we analyzed 72 enzymes involved in the DNA repair Mechanisms for their interactions with ligands (NNK and NNAL). The binding efficiencies with enzymes ranging from +36.96 to -7.47 Kcal/Mol. Crystal Structure of Human Carbonmonoxy-Haemoglobin at 1.25 Å Resolution, PDB ID-1IRD as a +Ve control, showed binding energy -6.31 to -6.68 Kcal/Mol. and Human heat shock factor-binding protein 1, PDB ID- 3CI9 as a -Ve control, showed - 3.91 to +2.09 Kcal/Mol. Binding was characterized for the enzymes sharing equivalent or better interaction as compared to +Ve control. Study indicated the loss of functions of these enzymes, which probably could be a reason for fettering of DNA repair pathways resulting in damage accumulation and finally cancer formation.     

A structural model for the rolA protein and its interaction with DNA

The study of the plant oncogene rolA has been hampered by a lack of structural information. Here we show that, despite a lack of significant sequence similarity to proteins of known structure, the rolA sequence adopts a known fold; that of the papillomavirus E2 DNA-binding domain. This fold is reliably identified by modern threading programs, which consider predicted secondary structure, but not by others. Although the rolA sequence is only around 16% identical to those of the available template structures, a structural model could be built that performed well against protein structure verification programs. The adopted strategy involved alignment corrections, justified by multiple model building and evaluation, with particular attention paid to the hydrophobic core residues. We find that rolA protein is predicted to resemble the template proteins in two key aspects; existence as a dimer and ability to bind DNA. rolA protein has recently been shown experimentally to possess DNA binding ability. This model predicts Lys 24 and Arg 27 to be involved in sequence-specific interactions and eight other residues to hydrogen-bond phosphate groups of the DNA.     

Interaction between second generation anthracyclines and DNA in the nucleosomal structure.

The interaction between three anthracycline antibiotics of second generation (9-deoxydoxorubicin, 9-DAM, 4-demethoxydaunorubicin, 4-DDM, 4'-deoxydoxorubicin, 4'-DAM) and DNA in the nucleosomal structure was investigated using fluorescence and circular dichroism techniques. The thermodynamic parameters of the binding process were obtained at different ionic strength and temperature conditions, thus allowing the calculation of the electrostatic contribution to the free energy and the enthalpy of the process. The same measurements were performed on linear double stranded DNA for comparison. The parent compounds adriamycin and daunomycin were additionally considered. Although the examined drugs greatly vary in biological activity, their binding parameters are only slightly different. Like the parent compounds, 9-DAM, 4-DDM and 4'-DAM exhibit preference for isolated regions of the polynucleotide rather than for nucleosomes. This fact suggests a non-homogeneous distribution of the antibiotics in vivo. The enthalpy values are remarkably lower than the ones characterizing the interaction of adriamycin and daunomycin to DNA. According to CD spectra, all derivatives, but 4-DDM, intercalate into nucleosomal or free DNA in a manner similar to the first generation compounds, namely with the chromophore perpendicular to the hydrogen bonds between the bases. The demethoxy compound, on the other hand, seems to be able to insert its planar moiety in different orientations, which are related to the structure of the nucleic acid being examined. The lack of the methoxy group in the intercalating part of the molecule appears to be responsible for this behaviour. As far as biological activity is concerned, our findings indicate a qualitative correlation between cell cytotoxicity and ability of interaction with nucleosomes at physiological conditions, rather than with free DNA. The modified binding stereochemistry of 4-DDM could play an additive role in modulating the pharmacological effectiveness of the above compounds.     

Molecular mechanical calculations on the interaction of ethidium cation with double-helical DNA

Molecular mechanical calculations were done on complexes of ethidium cation with various base-paired deoxydinucleoside monophosphates [(ApT)₂, (TpA)₂, (A₂ · T₂), (GpC)₂, (CpG)₂, and (G₂ · C₂)] and deoxyhexanucleoside pentaphosphates [(ATATAT)₂, (TATATA)₂, (A₆ · T₆), (GCGCGC)₂, (CGCGCG)₂, and G₆ · C₆]. Relative binding energies, sequence preferences, and conformational aspects of the intercalation complexes were studied. The most detailed models used (an all-atom force field) gave results in good agreement with previous calculations and experimental work. Less-sophisticated models did not perform as well.     

Molecular crowding regulates the structural switch of the DNA G-quadruplex

Almost all biochemical reactions in vitro have been investigated through numerous experiments conducted in dilute solutions containing low concentrations of solutes. However, biomacromolecules such as nucleic acids, proteins, and polysaccharides are designed to function and/or form their native structures in a living cell containing high concentrations of biomacromolecules, substrates, cofactors, salts, and so on. In the present study, we have demonstrated quantitatively the effect of molecular crowding on structures and stabilities of the G-quadruplex of d(G(4)T(4)G(4)). Molecular crowding with poly(ethylene glycol) (PEG) induced a structural transition from the antiparallel to the parallel G-quadruplex of d(G(4)T(4)G(4)), while molecular crowding with polycations did not alter the structure of the antiparallel G-quadruplex. The binding constants of putrescine, one of the polycations, for d(G(4)T(4)G(4)) in the absence and presence of Na(+) are calculated to be 277 and 2.5 M(-)(1), respectively. This indicates that the polycations coordinate to d(G(4)T(4)G(4)) with electrostatic interactions. The thermodynamic parameters of the antiparallel G-quadruplex formation under the crowding and noncrowding conditions induced by putrescine were also estimated. The stability of the antiparallel G-quadruplex decreased (-DeltaG degrees (25) decreased from 28 to 22 kcal mol(-)(1)) with molecular crowding by putrescine. Also, enthalpy and entropy changes in the structural formation under crowding and noncrowding conditions clearly showed that destabilization was entropy-driven. These quantitative parameters indicated that both the volume excluded by PEG and chemical interactions such as electrostatic interaction with solute polycations are critical for determining how molecular crowding affects the structure and stability of highly ordered DNA structures.     

Molecular cloning of Bacillus subtilis genes involved in DNA metabolism

Summary Different clones carrying a chromosomal DNA fragment able to transform Bacillus subtilis mutants dnaA13, dnaB19, dnaG5, recG40 and polA42 to a wild-type phenotype were isolated from a library constructed in plasmid pJH101. A recombinant clone carrying a chromosomal fragment able to transform dnaC mutants was obtained from a Charon 4A library. A restriction map of the cloned DNA fragments was constructed. The 11.3 kb cloned DNA fragment of plasmid pMP60-13 containing the wild-type sequence of dnaG5 was shown to transform a recF33 mutant as well.     

Molecular dynamics of structural transitions and intercalation in DNA

The large-scale flexibility of DNA and the intercalation of actinomycin D have been studied by computer simulation using molecular dynamics. The stretching and unwinding of B and Z forms of DNA and intercalation in B-DNA were examined through molecular dynamics simulations, and the energetics of transitions were calculated by the conformational energy minimization method. The principal results of this research are as follows: (1) A dynamic conformational pathway is presented for longitudinal stretching and unwinding of the double helix to open an intercalation site. (2) Large-scale transitions are possible in both B and Z forms of DNA through a conformationally allowed kinetic pathway. (3) The stretching and untwisting of a 5′(CG)3′ step is energetically more favorable than for a GC step in B-DNA. (4) The formation of an adjacent second cavity in B-DNA requires larger energy than the formation of the first cavity, affirming the neighbor-exclusion principle of intercalation. (5) Docking an intercalated actinomycin D in the stretched structure is shown to be geometrically and energetically feasible.     

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.     

Analysis of cooperativity and ion effects in the interaction of quinacrine with DNA

The interaction of quinacrine with calf thymus DNA was monitored at several different ionic strengths using spectrophotometric and equilibrium dialysis techniques. The binding results can be explained, assuming each base pair is a potential binding site, using a model containing two negative cooperative effects: (1) ligand exclusion at binding sites adjacent to a filled binding site and (2) ligand–ligand negative cooperativity at adjacent filled binding sites. The logarithm of the observed equilibrium constant (K_obs) determined by this model varies linearily with log[Na⁺], as predicted by the ion condensation theory for polyelectrolytes. When the log K_obs plot is correlated for sodium release by DNA in the intercalation conformational change, the predicted number of ion pairs between the ligand and DNA is approximately two, as expected for the quinacrine dication. Even though K_obs depends strongly on ionic strength, the ligand negative cooperativity parameter ω was found to be indpendent of ionic strength within experimental error. This finding is also in agreement with the ion condensation theory, which predicts a relatively constant amount of condensed counterion on the DNA double helix over this ionic strength range. Drugs would, therefore, experience a relatively constant ionic environment when complexed to DNA even though the ionic conditions of the solvent could change considerably.