Interaction of topotecan-a DNA topoisomerase I inhibitor-with dual-stranded polydeoxyribonucleotides. V. Topotecan is able to cause single- and double-strand breaks in ring superhelical DNA in the absence of enzyme

Temperature, concentration, and time dependence for the emergence of breaks in the sugar-phosphate backbone in a circular supercoiled DNA (scDNA) was studied for the first time in the presence of topotecan (TPT) and in the absence of human DNA topoisomerase I (topo I). Because TPT is a comptothecin (CPT) derivative, it is the first example for the ability of molecules of CPT family to cause double-stranded breaks in scDNA in the absence of the enzyme. The experiments were carried out in low ionic strength solutions (10 mM sodium cacodylate) at neutral pH (6.8). Incubation time necessary for the appearance of double-stranded breaks in scDNA in the presence of TPT correlated with the time of formation of strong TPT-DNA complex. A model was suggested for the complex composed of two crossed DNA duplexes bound through a bridge of two dimers of TPT lactone form. According to this model, two carbonyl groups of D rings of different TPT dimers form hydrogen bonds with 2-amino groups of guanines located in the neighboring base pairs of diverse strands of one of DNA duplexes. At the same time, two other carbonyl groups of D rings of TPT dimers form hydrogen bonds with 2-amino groups of guanines spaced five bp apart in the same strand of the second DNA duplex.     

Effect of p53 protein redox states on binding to supercoiled and linear DNA.

The binding of p53 to its DNA consensus sequence is modulated by the redox state of the protein in vitro. We have shown previously that reduced wild-type p53 binds strongly to supercoiled DNA (scDNA) regardless of the presence or absence of p53CON. Here we compare the effects of oxidation of p53 by azodicarboxylic acid bis[dimethylamide] (diamide) and other agents on p53 binding to p53CON and to scDNA. Oxidation decreases the binding of p53 to scDNA; however, under conditions where binding to p53CON in a DNA fragment is completely abolished, some residual binding to scDNA is still observed. Increasing the concentration of oxidized p53 confers minimal changes in p53 binding to both scDNA and p53CON. Reduction of the oxidized protein by dithiothreitol neither restores its binding to DNA nor to p53CON in DNA fragments. In the presence of excess zinc ions, oxidation of p53 is, however, reversible. We conclude that the irreversibility of p53 oxidation is due, at least in part, to the removal of intrinsic zinc from its position in the DNA binding domain accompanied by a conformational change of the p53 molecule after oxidation of the three cysteines to which the zinc ion is coordinated in the reduced protein.     

Evaluation of conformational changes in the supercoiled DNA of eukaryotic cells by direct nucleoid fluorimetry. I. The comparative information value of various methods of studying structural disorders in the supercoiled DNA in thymocytes and macrophages

A direct fluorimetric method is elaborated for studying conformational distortions in the scDNA of "nucleoids" of macrophages and thymocytes. The method is more advantageous compared to methods of viscosimetry and sedimentation.     

Study of mechanisms of electric field-induced DNA transfection. IV. Effects of DNA topology on cell uptake and transfection efficiency.

Electric parameters and solvent conditions are known to influence the efficiency of DNA transfection of cells by a pulsed electric field (PEF). A previous study (Neumann, E., M. Schaefer-Ridder, Y. Wang, and P. H. Hofschneider. 1982. EMBO (Eur. Mol. Biol. Organ.) J. 1:841-845) has indicated that DNA topology is also an important determinant. We report an investigation of the PEF induced uptake, stability, and expression of three different topological isomers, circular supercoiled (scDNA), circular relaxed (crDNA), and linearized (lnDNA) forms of the plasmid pBR322, by Escherichia coli strain JM105. Monomeric pBR322 prepared by the electroelution from an agarose gel was in the supercoiled form. Treatment of the scDNA with wheat germ topoisomerase I removed the superhelicity and the DNA assumed the relaxed circular form. Treatment of scDNA by a restriction endonuclease, EcoRI or Hind III, linearized the DNA. The MgCl2-dependent bindings of all three forms of DNA to the cell surface were indistinguishable. So was the PEF induced cell uptake. In contrast, the transfection efficiency (TE) for the scDNA and the crDNA were high (approximately 2 x 10(8) micrograms-1 DNA at neutral pH), whereas that for the lnDNA was approximately five orders of magnitude lower (less than 1 x 10(3) micrograms-1 DNA). Analysis by agarose gel electrophoresis indicated that the PEF loaded lnDNA was degraded by the host cell within 3 h. However, the loaded scDNA and the crDNA were stable and expressed in the cytoplasm. We conclude that first, the PEF induced DNA entry into E. coli did not depend on the topology of the DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polylysine-coated mica can be used to observe systematic changes in the supercoiled DNA conformation by scanning force microscopy in solution

The conformations of supercoiled (sc) DNA and linear DNA bound to polylysine (PL)-coated mica were investigated by scanning force microscopy (SFM) in solution. From the polymer statistical analysis of linear DNA, we could distinguish between re-arrangements or trapping of the DNA on the surface. Conditions of re-arrangements to an almost equilibrated state can be achieved at appropriate PL surface concentrations. We could show that the ability of re-arrangements depends on the salt concentration of the adsorption/imaging buffer. Comparing the statistical analysis of the linear DNA with SFM images of scDNA suggested that irregular scDNA conformations are formed under conditions of trapping, whereas plectonemic structures are favoured under conditions of surface re-arrangements. Salt-dependent changes in the scDNA conformation over the range of 10–100 mM NaCl, as characterised by the parameters writhe and the superhelix radius r, are observable only under conditions that enable surface re-arrangements. The measured values of writhe suggest that the scDNA loses approximately one-half of the supercoils during the binding to the surface. At the same time r increases systematically with decreasing writhe, thus the scDNA topology remains determined by the constraints on supercoiling during the binding to PL-coated mica.     

Interaction of Topotecan,DNA Topoisomerase I Inhibitor,with Double-Stranded Polydeoxyribonucleotides. 5. Topotecan is Capable of Producing Single- and Double-Strand Breaks in Circular Supercoiled DNA in the Absence of the Enzyme

A study was made of the temperature, concentration, and time dependences for the emergence of breaks in the sugar-phosphate backbone of a circular supercoiled DNA (scDNA) in the presence of a campto-thecin derivative topotecan (TPT) and in the absence of DNA topoisomerase I (topo I). The experiments were carried out in low ionic strength solutions (10 mM sodium cacodylate) at neutral pH (6.8). The incubation time necessary for the appearance of double-strand breaks in scDNA in the presence of TPT correlated with the time of formation of strong TPT–DNA complex. This is the first demonstration that molecules of the camptothecin family can cause double-strand breaks in scDNA in the absence of the enzyme. A model is suggested for the complex composed of two crossed DNA duplexes bound through a bridge of two dimers of the TPT lactone form. According to this model, two carbonyl groups of D rings of different TPT dimers form hydrogen bonds with 2-amino groups of guanines located in the neighboring base pairs of different strands of one DNA duplex. At the same time, two other carbonyl groups of D rings of TPT dimers form hydrogen bonds with 2-amino groups of guanines 5 bp apart in one and the same strand of the second DNA duplex.     

Catalytic transformations of supercoiled DNA as studied by flow linear dichroism technique

A catalytic turnover of supercoiled DNA (scDNA) transformation mediated by topoisomerases leads to changes in the linking number (Lk) of the polymeric substrate by 1 or 2 per cycle. As a substrate of the topoisomerization reaction it is chemically identical to its product; even a single catalytic event results in the quantum leap in the scDNA topology. Non-intrusive continuous assay to measure the kinetics of the scDNA topoisomerization was performed. The development of such a technique was hindered because of multiple DNA species of intermediate topology present in the reaction mixture. The interrelation of DNA topology, its hydrodynamics, and optical anisotropy enable us to use the flow linear dichroism technique (FLD) for continuous monitoring of the scDNA topoisomerization reaction. This approach permits us to study the kinetics of DNA transformation catalyzed by eukaryotic topoisomerases I and II, as well as mechanistic characteristics of these enzymes and their interactions with anticancer drugs. Moreover, FLD assay can be applied to any enzymatic reaction that involves scDNA as a substrate. It also provides a new way of screening drugs dynamically and is likely to be potent in various biomedical applications.     

Electron and scanning force microscopy studies of alterations in supercoiled DNA tertiary structure.

The configuration of supercoiled DNA (scDNA) was investigated by electron microscopy and scanning force microscopy. Changes in configuration were induced by varying monovalent/divalent salt concentrations and manifested by variation in the number of nodes (crossings of double helical segments). A decrease in the concentration of monovalent cations from 50 mM to approximately 1 mM resulted in a significant change of apparent configuration of negatively supercoiled DNA from a plectonemic form with virtually approximately 15 nodes (the value expected for molecules of approximately 3000 bp) to one or two nodes. This result was in good agreement with values calculated using an elastic rod model of DNA and salt concentration in the range of 5-50 mM. The effect did not depend on the identity of the monovalent cation (Na(+), K(+)) or the nature of the support used for electron microscopy imaging (glow-discharged carbon film, polylysine film). At very low salt concentrations, a single denatured region several hundred base-pairs in length was often detected. Similarly, at low concentrations of divalent cations (Mg(2+), Ca(2+), Zn(2+)), scDNA was apparently relaxed, although the effect was slightly dependent on the nature of the cation. Positively supercoiled DNA behaved in a manner different from that of its negative counterpart when the ion concentration was varied. As expected for these molecules, an increase in salt concentration resulted in an apparent relaxation; however, a decrease in salt concentration also led to an apparent relaxation manifested by a slight decrease in the number of nodes. Scanning force microscopy imaging of negatively scDNA molecules deposited onto a mica surface under various salt conditions also revealed an apparent relaxation of scDNA molecules. However, due to weak interactions with the mica surface in the presence of a mixture of mono/divalent cations, the effect occurred under conditions differing from those used for electron microscopy. We conclude that the observed changes in scDNA configuration are inherent to the DNA structure and do not reflect artifacts arising from the method(s) of sample preparation.     

Characterization of the ATPase activity of the Escherichia coli RecG protein reveals that the preferred cofactor is negatively supercoiled DNA

RecG is a member of the superfamily 2 helicase family. Its possible role in vivo is ATP hydrolysis driven regression of stalled replication forks. To gain mechanistic insight into how this is achieved, a coupled spectrophotometric assay was utilized to characterize the ATPase activity of RecG in vitro. The results demonstrate an overwhelming preference for negatively supercoiled DNA ((-)scDNA) as a cofactor for the hydrolysis of ATP. In the presence of (-)scDNA the catalytic efficiency of RecG and the processivity (as revealed through heparin trapping), were higher than on any other cofactor examined. The activity of RecG on (-)scDNA was not due to the presence of single-stranded regions functioning as loading sites for the enzyme as relaxed circular DNA treated with DNA gyrase, resulted in the highest levels of ATPase activity. Relaxation of (-)scDNA by a topoisomerase resulted in a 12-fold decrease in ATPase activity, comparable to that observed on both linear double-stranded (ds)DNA and (+)scDNA. In addition to the elevated activity in the presence of (-)scDNA, RecG also has high activity on model 4Y-substrates (i.e. chicken foot structures). This is due largely to the high apparent affinity of the enzyme for this DNA substrate, which is 46-fold higher than a 2Y-substrate (i.e. a three-way with two single-stranded (ss)DNA arms). Finally, the enzyme exhibited significant, but lower activity on ssDNA. This activity was enhanced by the Escherichia coli stranded DNA-binding protein (SSB) protein, which occurs through stabilizing of the binding of RecG to ssDNA. Stabilization is not afforded by the bacteriophage gene 32 protein, indicating a species specific, protein-protein interaction is involved. These results combine to provide significant insight into the manner and timing of the interaction of RecG with DNA at stalled replication forks.     

Unpaired bases in superhelical DNA: kinetic evidence

Kinetic analysis of the early, fast reaction of superhelical DNA with formaldehyde reveals that this region or regions is 56% “single strand like” in character. Hydrogen-tritium exchange studies coupled with other considerations show that this reaction is not due to a difference in conformational motility between form I and form II molecules, but is due to unpaired or weakly hydrogen bonded, localized region(s) of the form I allomorph of circular DNA.     

Minor groove dimeric bisbenzimidazoles inhibit <Emphasis Type="Italic">in vitro</Emphasis> DNA binding to eukaryotic DNA topoisomerase I

The potential of six dimeric bisbenzimidazoles bound to scDNA to inhibit eukaryotic DNA topoisomerase (topo-I) was studied chemically; the tested compounds differed in linker structure and length. All the compounds inhibited topo-I, DB(7) being the most efficient; its inhibitory activity in vitro was 50-fold higher than that of camptothecin. It is noteworthy that inhibitory properties of nearly all the tested compounds increased many times if they were preincubated with scDNA for three days.     

Fluctuations in superhelical DNA.

The effect of superhelicity on the base-pair opening probability and on the probability of occurrence of cruciform states in palindromic regions is theoretically treated. The calculations show that below the superhelix density value of -sigma=0.05 superhelicity does not appreciably affect the characteristics of DNA secondary structure fluctuations. In the range of physiological superhelix densities sigma (-sigma=0.05-0.09) the base-pair opening probability markedly increases. However, within this range of sigma the base-pairs are opened only transiently and permanently open regions are not formed. Permanently opened regions appear at higher negative superhelix densities (-sigma greater than 0.10). At the values of -sigma higher than 0.06 a cruciform structure in the palindromic region centred in position 3965 proves to be the most probable fluctuational disturbance in the 0x174 duplex DNA. Different experimental approaches used for probing the fluctuations in superhelical DNA have been analysed. The results suggest that most direct quantitative information can be derived from data on the nicking of closed DNA by single strand-specific endonucleases. Such data (Wang, 1974) accord with the results of theoretical calculations. Calculations show that, due to base-pair opening, the total free energy of superhelical DNA should depend parabolically on sigma only up to some critical value of sigma=sigmac. If negative superhelicity exceeds this critical value, which under physiological conditions proves to be -sigma=0.085, the free energy should increase linearly with -sigma. The biological role of supercoiling is discussed in the light of obtained results.     

Increased accessibility of bases in DNA upon binding of acridine orange.

Acridine orange (AO) forms 1:1 complexes with dsDNA which are insoluble in aqueous media, exhibit red luminescence, have minimal green luminescence and resemble complexes of AO with ss nucleic acids. During formation and/or dissociation of these complexes, accessibility of DNA bases to two conformational probes, formaldehyde and diethyl pyrocarbonate is increased, suggesting that the base pairing is destroyed and DNA at least partially denatured. Adriamycin and Ellipticine, but not Ethidium Bromide exert similar destabilizing effects. The results confirm our earlier predictions based on thermodynamic calculations that the double helix undergoes destabilization upon binding an intercalator characterized by high cooperativity in interaction with ss nucleic acids. Thus, the highly cooperative ligand binding to ss sections during the "breathing" of the polymer may progressively destabilize the adjacent ds structure.     

EVOLUTIONARY IMPLICATIONS OF DNA DIVERGENCE IN THE DROSOPHILA OBSCURA GROUP

Using DNA–DNA hybridization, we have determined the degree of single-copy DNA (scDNA) divergence among eight species of the Drosophila obscura group. These include Old World and New World species as well as members of two subgroups. Contrary to classical systematics, members of the affinis subgroup are more closely related to American members of the obscura subgroup than are Old World species. The Old World species are not a monophyletic group. The degree of scDNA divergence among species is not necessarily correlated with morphology, chromosomal divergence, or ability to form hybrids. A unique pattern of hybrid formation was found: species separated by a ΔT_m of 6.5°C can form hybrids whereas species separated by a ΔT_m of 2.5°C cannot. As with other groups of Drosophila, the obscura group has discrete parts of the genome evolving at very different rates. The slow evolving fraction of the nuclear genome is evolving at about the same rate as mitochondrial DNA. The additional scDNA divergence accompanying the step from partial reproductive isolation (between North American pseudoobscura and the isolated Bogotà population) to full isolation is very small. The resolution of the technique was challenged by five closely related taxa with a maximum ΔT_m of 2.5°C separating them; the taxa were unambiguously resolved and the “correct” phylogeny recovered. Finally, there is some indication that scDNA in the obscura group may be evolving considerably slower than in the melanogaster subgroup.     

Nature of conformational restrictions in nuclear superhelical DNA from thymus lymphocytes

The lymphocyte nucleoids of mouse thymus contain about 40% of rapidly labelled nuclear RNA, about 9% of total intracellular protein and all nuclear DNA. Relaxation of superhelical DNA after thymocyte nucleoids treatment with pronase or RNAase suggests that non-histone proteins and/or RNAs are involved in conformational restrictions in the superhelical domains of cell DNAs. Thymocyte nucleoids proteins are represented by two groups of nonhistone proteins with molecular weights of 50 000-60 000 and 75 000-85 000. An essential role in the appearance of conformational restrictions in thymocyte superhelical DNA belongs to disulfide bonds.