The structure of 4-way DNA junctions: specific binding of bis-intercalators with rigid linkers.

During replication and recombination, two DNA duplexes lie side by side. We have developed reagents that might be used to probe structure during these critical processes; they contain two intercalating groups connected by a rigid linker that forces those groups to point in opposite directions. If their stereochemistry proves appropriate, such structure-specific agents should intercalate specifically into adjacent duplexes in the Y- and X-shaped structures (i.e. 3- and 4-way junctions, now known as 3H and 4H junctions) found at replication and recombination sites. We prepared DNA structures in which four duplexes were arranged in all possible combinations around 2- and 4-way junctions and then probed the accessibility to DNase I of all their phosphodiester bonds. In the absence of any bis-intercalators, 7-9 nucleotides (nt) in each of the strands in 4-way junctions were protected from attack; protected regions were significantly offset to the 3' side of the junction in continuous strands, but only slightly offset, if at all, in exchanging strands. All the intercalators decreased accessibility throughout the structure, but none did so at specific points in the two adjacent arms of 4-way junctions. However, one bis-intercalator--but not its sister with a shorter linker--strikingly increased access to a particular CpT bond that lay 9 nt away from the centre of some 4-way junctions without reducing access to neighbouring bonds. Binding was both sequence and structure specific, and depended on complementary stereochemistry between bis-intercalator and junction.     

Selective recognition of pyrimidine-pyrimidine DNA mismatches by distance-constrained macrocyclic bis-intercalators

Binding of three macrocyclic bis-intercalators, derivatives of acridine and naphthalene, and two acyclic model compounds to mismatch-containing and matched duplex oligodeoxynucleotides was analyzed by thermal denaturation experiments, electrospray ionization mass spectrometry studies (ESI-MS) and fluorescent intercalator displacement (FID) titrations. The macrocyclic bis-intercalators bind to duplexes containing mismatched thymine bases with high selectivity over the fully matched ones, whereas the acyclic model compounds are much less selective and strongly bind to the matched DNA. Moreover, the results from thermal denaturation experiments are in very good agreement with the binding affinities obtained by ESI-MS and FID measurements. The FID results also demonstrate that the macrocyclic naphthalene derivative BisNP preferentially binds to pyrimidine–pyrimidine mismatches compared to all other possible base mismatches. This ligand also efficiently competes with a DNA enzyme (M.TaqI) for binding to a duplex with a TT-mismatch, as shown by competitive fluorescence titrations. Altogether, our results demonstrate that macrocyclic distance-constrained bis-intercalators are efficient and selective mismatch-binding ligands that can interfere with mismatch-binding enzymes.     

Quantitative and sequence-specific analysis of DNA-ligand interaction by means of fluorescent intercalator probes

A novel method of analysis of double-stranded DNA-ligand interaction is presented. The interaction is monitored by the fluorescence of a DNA bis-intercalator oxazole homodimer YoYo-3. The fluorescence intensity or its decay time reflects the modification of the DNA double helix. The DNA sequence is scanned by hybridization with short oligomers having consecutively overlapping complementary sequences to analyse the sequence specificity of binding. In our experiments we used as ligands the minor groove binders netropsin, SN6999 (both with AT-preference), the GC-specific ligand chromomycin A3 as well as the derivative SN6113 (non-specific interaction), which displace the bis-intercalator YoYo-3 or influence the duplex structure in such away that the fluorescence intensity and lifetime decrease in comparison to a ligand-free screening. The changes of fluorescence emission clearly define the binding motif and indicate minor groove interactions with a reduced DNA binding site. Titration of the ligand quantitatively characterizes its binding by determining the dependence of the binding constant on the oligonucleotide sequence.     

Analysis of the effects of antibiotics on the structure of nucleosome core particles determined by DNAase I cleavage

Patterns of sensitivity to DNAase I cleavage have been analysed in order to investigate the effects of anti-tumour antibiotics and related drugs on nucleosome core particles containing different DNA restriction fragments. In this article, we review the experimental results which show that after controlled digestion of defined-sequence core particles, new cleavage products appear in the enzyme digestion patterns which lie approximately mid-way between the strong bands characteristic of native nucleosome core particles. The effects of the antibiotics, which include bis-intercalators as well as minor groove-binding ligands (but not monofunctional intercalators), are explained in terms of an induced change in rotational setting (phasing) of the core DNA. The new rotational positioning of DNA induced by antibiotic binding appears to be almost independent of DNA sequence, although some differences can be observed with the various pieces of DNA, most likely reflecting the exact number and disposition of antibiotic binding sites.     

Bis-intercalative binding to DNA of novel bis(10-methyl)acridinium chlorides and its dependence on chain length of linker.

The synthesis of a series of novel bis(10-methyl)acridinium compounds (both unsubstituted and the 6-chloro-2-methoxy substituted) linked by methylene bridges of lengths from (CH2)4 to (CH2)12 and in one case by spermine is described. Their ability to bind to duplex DNA was compared by their relative inhibition of E. coli DNA polymerase catalyzed DNA synthesis. It was determined that they function as DNA template inhibitors and do not affect the DNA polymerase directly. Their ability to function as bis-intercalators was assessed by a novel and convenient topoisomerase fluorescent assay. It was concluded that whereas the (CH2)4-linked compounds act only as monofunctional intercalators because of steric constraints the (CH2)6-, (CH2)8-, and (CH2)10-linked substituted bisacridinium compounds, as well as the (CH2)10- and (CH2)12- unsubstituted analogues, function as bis-intercalators with DNA.     

DNA triple helix stabilisation by a naphthylquinoline dimer

We have used DNase I footprinting to examine the effect of a novel naphthylquinoline dimer, designed as a triplex-specific bis-intercalator, on the stability of intermolecular DNA triplexes. We find that this compound efficiently promotes triplex formation between the 9-mer oligonucleotide 5'-TTTTTTCTT and its oligopurine duplex target at concentrations as low as 0.1 microM, enhancing the triplex stability by at least 1000-fold. This compound, which is the first reported example of a triplex bis-intercalator, is about 30 times more potent than the simple monofunctional ligand.     

The association of penicillin-binding proteins with cell elongation and septum formation in Bacillus megaterium.

The synthesis of a new bifunctional compound in which two aminoacridine chromophores are linked by the bicyclic depsipeptidic backbone of des-N-tetramethylTriostin A is described. The molecule, bis-[(9-acridinyl)-D-seryl-L-alanyl-L-cysteinyl-L-valine] dilactone disulphide, structurally analogous to the antibiotic anti-tumour drug Triostin A, is shown to possess a high affinity to DNA and to act as a bis-intercalator on the basis of spectroscopic, viscosimetric and thermal-denaturation studies. This model constitutes the first attempt of a synergic association between a peptidic moiety that mimics a naturally occurring drug and aminoacridine, the two parts themselves each exhibiting a high affinity for the DNA target.     

DNA repair synthesis induced by N-hydroxyurea, acetohydroxamic acid, and N-hydroxyurethane in primary rat hepatocyte cultures: comparative evaluation using the autoradiographic and the bromodeoxyuridine density-shift method

N-Hydroxyurea and two structurally related compounds, acetohydroxamic acid and N-hydroxyurethane, were investigated for their potential to induce DNA repair synthesis in primary rat hepatocyte cultures. Repair was determined as repair replication by means of the bromodeoxyuridine density-shift method and, in the same cell preparations, as unscheduled DNA synthesis (UDS) by autoradiography. For all 3 compounds, a clear concentration-dependent induction of DNA repair replication could be demonstrated. Interpretation of the UDS data, however, depended on the mode whereby the results were evaluated. Expression of the results as net grains per nucleus after subtraction of cytoplasmic from nuclear grain counts yielded statistically significant increases over the control values for all compounds. In contrast, no significant changes of the nuclear labeling were obtained when nuclear and cytoplasmic grain counts were plotted separately. These findings demonstrate that the two modes to present UDS data may lead to different conclusions, a consequence of the uncertainty regarding the origin and importance of the cytoplasmic background. The observation that both hydroxyurea and the structurally related compounds acetohydroxamic acid and N-hydroxyurethane induce DNA repair in primary hepatocyte cultures suggests that metabolism-dependent genotoxicity may be a common property of aliphatic hydroxamic acids.     

Inhibition of DNA replication in Escherichia coli by dibromophenol and other uncouplers.

DNA replication in Escherichia coli is inhibited by uncouplers such as 2,4-dibromophenol and 3,3'4',5-tetrachlorosalicylanilide. Inhibition occurs in either aerobically or anaerobically growing cells or in cells made permeable by toluene. With anaerobically growing cells, inhibition by dibromophenol is reversible and occurs under conditions in which there is no change in pools of ATP or deoxynucleoside triphosphates. With toluenized cells, inhibition is not due to breakdown of deoxynucleoside triphosphates. The rates of protein and RNA synthesis are not inhibited either in vivo or in toluenized cells by concentrations of dibromophenol or tetrachlorosalicylanilide which inhibit replication. It is generally believed that uncouplers inhibit many other cellular processes by collapsing a proton gradient across a membrane. However, the relative effectiveness of eight uncouplers and related compounds to inhibit replication did not parallel their ability to transport protons into E. coli cells. Therefore, the inhibition by uncouplers does not suggest that replication depends on a chemiosmotic process. A possible explanation for the uncoupler sensitivity is provided by the finding that many of the purified enzymes tested, including DNA polymerases II and III, are inhibited by dibromophenol and tetrachlorosalicylanilide.     

Structural perturbations in DNA caused by bis-intercalation of ditercalinium visualised by atomic force microscopy

Atomic force microscopy (AFM) has been used to examine perturbations in the tertiary structure of DNA induced by the binding of ditercalinium, a DNA bis-intercalator with strong anti-tumour properties. We report AFM images of plasmid DNA of both circular and linearised forms showing a difference in the formation of supercoils and plectonemic coils caused at least in part by alterations in the superhelical stress upon bis-intercalation. A further investigation of the effects of drug binding performed with 292 bp mixed-sequence DNA fragments, and using increment in contour length as a reliable measure of intercalation, revealed saturation occurring at a point where sufficient drug was present to interact with every other available binding site. Moment analysis based on the distribution of angles between segments along single DNA molecules showed that at this level of bis-intercalation, the apparent persistence length of the molecules was 91.7 ± 5.7 nm, approximately twice as long as that of naked DNA. We conclude that images of single molecules generated using AFM provide a valuable supplement to solution-based techniques for evaluation of physical properties of biological macromolecules.     

An image-based, high-throughput screening assay for molecules that induce excess DNA replication in human cancer cells

Previous studies have shown DNA re-replication can be induced in cells derived from human cancers under conditions in which it is not possible for cells derived from normal tissues. Because DNA re-replication induces cell death, this strategy could be applied to the discovery of potential anticancer therapeutics. Therefore, an imaging assay amenable to high-throughput screening was developed that measures DNA replication in excess of four genomic equivalents in the nuclei of intact cells and indexes cell proliferation. This assay was validated by screening a library of 1,280 bioactive molecules on both normal and tumor-derived cells where it proved more sensitive than current methods for detecting excess DNA replication. This screen identified known inducers of excess DNA replication, such as inhibitors of microtubule dynamics, and novel compounds that induced excess DNA replication in both normal and cancer cells. In addition, two compounds were identified that induced excess DNA replication selectively in cancer cells and one that induced endocycles selectively in cancer cells. Thus, this assay provides a new approach to the discovery of compounds useful for investigating the regulation of genome duplication and for the treatment of cancer.     

Repair replication of opossum lymphocyte DNA: effect of compounds that bind to DNA

Opossum lymphocytes were used for studies of DNA repair. Several compounds were assessed for their capacity to induce repair. Specially interesting was the fact that some intercalators (proflavin, ICR-170, quinacrine and acridine orange) did induce repair, as determined by [³H]thymidine incorporation in the presence of hydroxyurea, CsCl density gradient centrifugation of bromodeoxyuridine-containing DNA and autoradiographically detected unscheduled DNA synthesis.A comparison of the inhibitory effect of several chemicals on DNA replication and DNA repair was also carried out. In this study, repair synthesis was induced by UV irradiation. For most of the compounds, the concentration necessary to inhibit 50% of DNA replication or DNA repair was similar. The most notable exception was cycloheximide which inhibited replication much more effectively than repair. None of the compounds used in this study was found to specifically inhibit repair synthesis.Inhibition of DNA replication and DNA repair was a general effect exhibited by the compounds which bind to DNA. However, only some of these compounds were able to induce repair. As most of these compounds were mutagens it was concluded that the inhibitory effect could be more relevant to mutagenesis that the repair-induction effect.     

Translesion replication by DNA polymerase beta is modulated by sequence context and stimulated by fork-like flap structures in DNA

Mutations in the human genome are clustered in hot-spot regions, suggesting that some sequences are more prone to accumulate mutations than others. These regions are therefore more likely to lead to the development of cancer. Several pathways leading to the creation of mutations may be influenced by the DNA sequence, including sensitivity to DNA damaging agents, and repair mechanisms. We have analyzed sequence context effects on translesion replication, the error-prone repair of single-stranded DNA regions carrying lesions. By using synthetic oligonucleotides containing systematic variations of sequences flanking a synthetic abasic site, we show that translesion replication by the repair polymerase DNA polymerase beta is stimulated to a moderate extent by low stacking levels of the template nucleotides downstream of the lesion, combined with homopolymeric runs flanking the lesion both upstream and downstream. A strong stimulation of translesion replication by DNA polymerase beta was seen when fork-like flap structures were introduced into the DNA substrate downstream of the lesion. Unlike for gapped substrates, this stimulation was independent of the presence of a phosphate group at the 5' terminus of the flap. These results suggest that DNA polymerase beta may participate in cellular DNA transactions involving higher order structures. The significance of these results for in vivo translesion replication is discussed.     

Mutation induction in Haemophilus influenzae by ICR-191 II. Role of DNA replication and repair

Evidence is presented to show that presumptive frameshift mutations induced in Haemophilus influenzae by ICR-191 are fixed very rapidly, essentially at the time of treatment. DNA synthesis during treatment is essential for fixation, but DNA synthesis after treatment has no effect. The conclusion is drawn that the mutagen acts at the replication fork, possibly to stabilize misannealings arising in association with the discontinuities in the newly synthesized DNA. These results agree with earlier results on Escherichia coli showing that ICR-191 produces peak mutation frequencies in synchronized cultures at times when the replication fork has reached the locus being studied. They are in sharp contrast to the earlier results in H. influenzae with nitroso compounds and hydrazine that suggest these agents produce randomly distributed, reparable pre-mutational damage that still can be fixed (converted to final mutation) for some time after treatment when the replication fork reaches them. No evidence for such persistent pre-mutational lesions was found with ICR-191. A defect in incision appeared to have very little influence on mutation induction by ICR-191 though it caused much more lethality. The interpretation of the mutation data was made somewhat uncertain, however, by an unexplained plating-density effect on the expression of the mutants in this strain. In contrast, incision deficiencies in E. coli and Salmonella typhimurium have been reported to cause a large increase in mutation induction and to allow lesions at some distance from the replication fork to produce mutations.     

Receptor independent effects on DNA replication by steroids

There is now convincing evidence associating estrogens with an increased risk of some cancers. However, the absence of a complete correlation between estrogen receptor binding and the biological activity of these estrogens has suggested the possibility of other mechanisms of action. The effect on DNA replication of several hormones that are putatively involved in breast cancer was tested at a physiological concentration. The studies were conducted in a HeLa cell-free system by using a plasmid containing a specific mammalian origin of replication (DHFR oriβ<0R) as template DNA. A series of related steroids produced an entire range of activity from enhancement to inhibition of in vitro DNA replication. These studies indicate a new possible target, which may help to better understand the effect of these hormones in breast cancer. Furthermore, the results show that this in vitro DNA replication system provides an evaluative assay for the effects of compounds on hormone-responsive cancers independent of some hormone receptors. J. Cell. Biochem. 70:323–329, 1998. © 1998 Wiley-Liss, Inc.     

Inhibition of Epstein-Barr virus replication by a benzimidazole L-riboside: novel antiviral mechanism of 5, 6-dichloro-2-(isopropylamino)-1-beta-L-ribofuranosyl-1H-benzimidazole.

Although a number of antiviral drugs inhibit replication of Epstein-Barr virus (EBV) in cell culture, and acyclovir (ACV) suppresses replication in vivo, currently available drugs have not proven effective for treatment of EBV-associated diseases other than oral hairy leukoplakia. Benzimidazole riboside compounds represent a new class of antiviral compounds that are potent inhibitors of human cytomegalovirus (HCMV) replication but not of other herpesviruses. Here we characterize the effects of two compounds in this class against lytic replication of EBV induced in a Burkitt lymphoma cell line latently infected with EBV. We analyzed linear forms of EBV genomes, indicative of lytic replication, and episomal forms present in latently infected cells by terminal probe analysis followed by Southern blot hybridization as well as the high-molecular-weight unprocessed viral DNA by pulsed-field gel electrophoresis. D-Ribofuranosyl benzimidazole compounds that act as inhibitors of HCMV DNA maturation, including BDCRB (5, 6-dichloro-2-bromo-1-beta-D-ribofuranosyl-1H-benzimidazole), did not affect the accumulation of high-molecular-weight or monomeric forms of EBV DNA in the induced cells. In contrast, the generation of linear EBV DNA as well as precursor viral DNA was sensitive to the L-riboside 1263W94 [5, 6-dichloro-2-(isopropylamino)-1-beta-L-ribofuranosyl-1H-benzimidazole]. The 50% inhibitory concentration range for 1263W94 was 0.15 to 1. 1 microM, compared with 10 microM for ACV. Thus, 1263W94 is a potent inhibitor of EBV. In addition, 1263W94 inhibited the phosphorylation and the accumulation of the essential EBV replicative cofactor, early antigen D.     

The mechanism of inhibition of DNA replication in HeLa S3 cells by the antitumor drug Ledakrin and other antitumor 1-nitro-9-aminoacridines

These studies were aimed at characterizing the capability of an antitumor DNA-damaging drug, Ledakrin, and its analogs to inhibit DNA replication in HeLa S3 cells. The studied agents are extremely potent inhibitors of [³H]thymidine incorporation in whole cells. These compounds produced also a potent dose- and time-dependent inhibition of DNA synthesis in subcellular systems derived from drug-treated cells, as found by [³H]dGTP incorporation in cellular lysates and nuclei. Experiments in which nuclei from control and drug-treated cells were supplemented with cytoplasmic fractions from either control or drug-treated cells, or with exogenous DNA, demonstrate that Ledakrin and other 1-nitro-9-aminoacridines inhibit DNA replication in HeLa S3 cells by interfering with the DNA template, while not affecting DNA polymerase(s) or other enzymes and replication factors. The negligible effect of Ledakrin added to lysates or nuclei from untreated cells suggests that metabolic activation is a prerequisite for replication inhibition by Ledakrin. Analysis of the size of newly synthesized DNA, by alkaline sucrose gradient sedimentation, indicates that Ledakrin does not inhibit the initiation of replication but does interfere with chain growth. Impairment of DNA replication by 1-nitro-9-aminoacridines seems to originate from DNA damage and to result in the inhibition of cellular growth.     

Analogs of natural nucleosides and their 5'-triphosphates carrying the amino group in 3'-position--highly effective inhibitors of replication, repair and transcription

The new type of DNA and RNA synthesis inhibitors is found. These compounds are active in synthesis, catalyzed by bacterial DNA and RNA polymerases and by eukaryotic DNA polymerases in vitro and in DNA replication in vivo.