Reactivity of monofunctional cis-platinum adducts as a function of DNA sequence.

The purpose of this work was to study the chemical reactivity of monofunctional cis-platinum-nucleic acid adducts as a function of nucleic acid sequence. The first part of the paper deals with the formation of these adducts. It is shown that the ternary nucleic acid-cis-platinum-ethidium bromide complexes in which ethidium bromide and nucleotide residues are cross-linked by cis-platinum, are relatively unstable at 37 degrees C. In the presence of acridine, ethidium bromide (but not cis-platinum) is slowly released which leads to the formation of monofunctional cis-platinum-nucleic acid adducts. After removal of acridine, the monofunctional adducts react further to become bifunctional. The second part of the paper deals with the kinetics of disappearance of the monofunctional adducts in several polynucleotides but not in poly(dG).poly(dC). When the adducts possess a chloride ligand, the limiting step in the cross-linking is the rate of aquation reaction of the chloride ligand. The rate constants are an order of magnitude larger when the monofunctional adducts do not possess a chloride ligand. In both the cases, the rate constants are apparently independent of the nucleic acid sequence.     

Distortions induced in DNA by cis-platinum interstrand adducts.

A 22 base pair double-stranded oligonucleotide containing a unique interstrand adduct resulting from chelation of the two guanine residues within the central sequence d(TGCT/AGCA) by a cis-platinum residue has been studied by means of gel electrophoresis, chemical probes, and molecular mechanics. The anomalously slow electrophoretic mobility of the multimers of the platinated and ligated oligomers suggests that the platinated oligonucleotide is bent. The two cytosine residues (complementary to the platinated guanines) are hyperreactive to hydroxylamine, indicating a large exposure of the two bases to the solvent. The adduct does not induce a local denaturation within the flanking sequences since the adenine residues are not reactive with diethyl pyrocarbonate. This is confirmed by the nonreactivity of the complementary T residues with osmium tetraoxide. These results and the molecular mechanics modeling suggest that the interstrand adduct bends the double helix by approximately 55 degrees toward the major groove, that the double helix conserves its average twist angle, and that the distortion induced by the adduct is localized at the platinated sequence d(GC/CG).     

Formation of a DNA monofunctional cis-platinum adduct cross-linking the intercalating drug N-methyl-2,7-diazapyrenium.

Our purpose was to better understand the mutual influence of cis-diamminedichloroplatinum (II) (cis-DDP) and intercalating drugs in their interactions with DNA. The present study deals with the intercalating drug N-methyl-2,7-diazapyrenium (MDAP). Two sets of experiments have been performed. In one set, the reaction between cis-DDP and nucleic acid was carried out in the presence of MDAP. The main adduct is a guanine residue chelated by platinum to a MDAP residue. It has the same spectroscopic properties as the synthesized compound cis-[Pt (NH3)2 (N7-d-guanosine) (N7-MDAP)] , the structure of which has been determined by 1H NMR. This adduct was only formed with double-stranded nucleic acids which reveals the importance of DNA matrix in orienting favorably the reactants. In the second set of experiments, the triamine complex cis-[Pt(NH3)2 (MDAP)CI]++ was reacted with the nucleic acids. At molar ratios drug over nucleotide residue equal or less than 0.10, all the added triamine complexes bind by covalent coordination to double-stranded nucleic acids. With natural DNA, the major adduct is cis-[Pt(NH3)2(d-guanosine) (MDAP)] . Thus the same adduct is formed on one hand in the reaction between DNA, MDAP and cis-DDP and on the other hand in the reaction between the triamine complex and DNA. The triamine complex offers the possibility to study the biological role of the new adduct.     

The flexible DNA double helix. II. Superhelix formation.

A simple super or s-virtual bond scheme has been developed for the treatment of tertiary or superhelical structure in polynucleotide chains. The various spatial configurations accessible to the flexible double helix are rendered more readily intelligible by the introduction of these hypothetical bonds to replace real sequences of regular secondary structure. The scheme is utilized to examine the enormous variety of tertiary structure that can be generated by regularly bending a B-DNA reference helix at the phosphodiester linkages. Of particular interest from the study are the large families of bends that generate superhelices of identical macroscopic dimensions. Various modes of folding the B-type helix into superhelices that fit the experimentally measured dimensions of chromatin nucleosomes are illustrated.     

Chemical reactivity of monofunctional platinum-DNA adducts

Complexes formed in vitro between cis- or trans-PtCl2(NH3)2 (DDP) and DNA were found to contain monofunctional adducts that reacted with exogenous guanosine. [14C]Guo bound irreversibly to cis- and trans-DDP-DNA complexes to form bis-Gua adducts. The reaction was first order with respect to the concentration of both [14C]Guo and platinum-DNA complex, but the rate of the reaction varied nonlinearly as a function of the level of platinum binding on DNA. The reaction between [14C]Guo and these platinum-DNA complexes was used to probe the concentration and stability of the monofunctional adducts and to investigate their chemistry in situ. The concentration of monofunctional adducts was highest immediately after reaction of DDP with DNA for 2 h at 37 degrees C, at which time they represented greater than 15% of the cis-DDP-DNA lesions and on the order of 80% of the trans-DDP-DNA lesions. The cis-DDP-DNA complex reacted with [14C]Guo by two kinetically distinct processes, indicating two types of reactive adducts. The most reactive adduct represented 5% of the platinum lesions. These monofunctional adducts disappeared during the incubation of the platinum-DNA complexes in the absence of drug, probably as a result of chelation to DNA. The half-lives of this chelation at 37 degrees C, 10 mM NaClO4, were 15 and 30 h for the cis and trans complexes, respectively. Monofunctional adducts were formed on Gua bases in DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Strong bending of the DNA double helix

During the past decade, the issue of strong bending of the double helix has attracted a lot of attention. Here, we overview the major experimental and theoretical developments in the field sorting out reliably established facts from speculations and unsubstantiated claims. Theoretical analysis shows that sharp bends or kinks have to facilitate strong bending of the double helix. It remains to be determined what is the critical curvature of DNA that prompts the appearance of the kinks. Different experimental and computational approaches to the problem are analyzed. We conclude that there is no reliable evidence that any anomalous behavior of the double helix happens when DNA fragments in the range of 100 bp are circularized without torsional stress. The anomaly starts at the fragment length of about 70 bp when sharp bends or kinks emerge in essentially every molecule. Experimental data and theoretical analysis suggest that kinks may represent openings of isolated base pairs, which had been experimentally detected in linear DNA molecules. The calculation suggests that although the probability of these openings in unstressed DNA is close to 10⁻⁵, it increases sharply in small DNA circles reaching 1 open bp per circle of 70 bp.     

Tau could protect DNA double helix structure

The hyperchromic effect has been used to detect the effect of tau on the transition of double-stranded DNA to single-stranded DNA. It was shown that tau increased the melting temperature of calf thymus DNA from 67 to 81 degrees C and that of plasmid from 75 to 85 degrees C. Kinetically, rates of increase in absorbance at 260 nm of DNA incubated with tau were markedly slower than those of DNA and DNA/bovine serum albumin used as controls during thermal denaturation. In contrast, rates of decrease in the DNA absorbance with tau were faster than those of controls when samples were immediately transferred from thermal conditions to room temperature. It revealed that tau prevented DNA from thermal denaturation, and improved renaturation of DNA. Circular dichroic spectra results indicated that there were little detectable conformational changes in DNA double helix when tau was added. Furthermore, tau showed its ability to protect DNA from hydroxyl radical (.OH) attacking in vitro, implying that tau functions as a DNA-protecting molecule to the radical.     

Force-induced melting of the DNA double helix 1. Thermodynamic analysis

The highly cooperative elongation of a single B-DNA molecule to almost twice its contour length upon application of a stretching force is interpreted as force-induced DNA melting. This interpretation is based on the similarity between experimental and calculated stretching profiles, when the force-dependent free energy of melting is obtained directly from the experimental force versus extension curves of double- and single-stranded DNA. The high cooperativity of the overstretching transition is consistent with a melting interpretation. The ability of nicked DNA to withstand forces greater than that at the transition midpoint is explained as a result of the one-dimensional nature of the melting transition, which leads to alternating zones of melted and unmelted DNA even substantially above the melting midpoint. We discuss the relationship between force-induced melting and the B-to-S transition suggested by other authors. The recently measured effect on T7 DNA polymerase activity of the force applied to a ssDNA template is interpreted in terms of preferential stabilization of dsDNA by weak forces approximately equal to 7 pN.     

DNA self-fitting: the double helix directs the geometry of its supramolecular assembly.

Groove-backbone interaction is a natural and biologically relevant mechanism for the specific assembly of B-DNA double helices. Crystal engineering and crystal packing analysis of oligonucleotides of different sizes and sequences reveal that the sequence-dependent self-fitting of B-DNA helices is a dominant constraint for their ordered assembly. It can override the other intermolecular interactions and impose the overall geometry of the packing. Analysis of experimental examples of architectural motifs formed by the geometric combination of self-fitted DNA segments leads to general rules for DNA assembly. Like a directing piece for a supramolecular 'construction set', the double helix imposes a limited number of geometric solutions. These basic architectural constraints could direct, in a codified manner, the formation of higher-order structures. DNA architectural motifs exhibit new structural and electrostatic properties which could have some implications for their molecular recognition by proteins acting on DNA.     

Predicted role of particular phonons in the strand separation melting of the DNA double helix

The frequency-dependent vibrational fluctuation of the hydrogen bonds around a nucleation defect for the strand separation melting of a DNA polymer poly(dG)-poly(dC) is studied using a modified self-consistent phonon theory. There are two critical frequency bands around the defect at 340 K which is near the temperature at which the hydrogen bonds in neighboring cells melt. The first band is between 60 cm-1 and 120 cm-1 which is essential for the melting proceeding in +z direction(3'----5' in the G backbone). The second is the band under 20 cm-1 which is important for the melting proceeding in -z direction.     

Statistical-mechanical treatment of violations of the double helix in supercoiled DNA.

We consider the problem of making allowance for superhelicity in the statistical-mechanical calculations of fluctuational violations of the DNA double helix. A simple model is discussed, making it possible in the calculations to use an approach based on the theory of helix–coil transition in DNA. The proposed algorithms allow calculating the effect of superhelicity on the base-pair fluctuational opening for any given sequence of nucleotides. An algorithm is also proposed allowing for the hairpin and cruciform structures in the palindromic regions of a sequence, as well as the open and helical states. The theory is used to calculate the melting curve for superhelical DNA at temperatures well below the melting point of the linear or nicked forms. The maps of opening probability are calculated for SV40 and ?X174 DNA using their recently published complete nucleotide sequences. The data explain well the experimental results of probing the secondary structure of these DNA by single strand-specific endonucleases.     

Sequence-dependent base pair opening in DNA double helix

Preservation of genetic information in DNA relies on shielding the nucleobases from damage within the double helix. Thermal fluctuations lead to infrequent events of the Watson-Crick basepair opening, or DNA "breathing", thus making normally buried groups available for modification and interaction with proteins. Fluctuational basepair opening implies the disruption of hydrogen bonds between the complementary bases and flipping of the base out of the helical stack. Prediction of sequence-dependent basepair opening probabilities in DNA is based on separation of the two major contributions to the stability of the double helix: lateral pairing between the complementary bases and stacking of the pairs along the helical axis. The partition function calculates the basepair opening probability at every position based on the loss of two stacking interactions and one base-pairing. Our model also includes a term accounting for the unfavorable positioning of the exposed base, which proceeds through a formation of a highly constrained small loop, or a ring. Quantitatively, the ring factor is found as an adjustable parameter from the comparison of the theoretical basepair opening probabilities and the experimental data on short DNA duplexes measured by NMR spectroscopy. We find that these thermodynamic parameters suggest nonobvious sequence dependent basepair opening probabilities.     

Cytosine, the double helix and DNA self-assembly

DNA self-assembly has crucial implications in reading out the genetic information in the cell and in nanotechnological applications. In a recent paper, self-assembled DNA crystals displaying spectacular triangular motifs have been described (Zheng et al., 2009). The authors claimed that their data demonstrate the possibility to rationally design well-ordered macromolecular 3D DNA lattice with precise spatial control using sticky ends. However, the authors did not recognize the fundamental features that control DNA self-assembly in the lateral direction. By analysing available crystallographic data and simulating a DNA triangle, we show that the double helix geometry, sequence-specific cytosine–phosphate interactions and divalent cations are in fact responsible for the precise spatial assembly of DNA.