Depletion of intracellular polyamines may alter DNA conformation in 9L rat brain tumor cells.

Depletion of polyamines in 9L rat brain tumor cells by treatment with alpha-difluoromethylornithine dramatically altered DNA conformation as measured by viscoelastometry. The reduction of intracellular putrescine and spermidine concentrations to less than 5 percent of their concentrations in control cells decreased the sensitivity of 9L cell DNA to x-irradiation and increased the maximum viscoelastic retardation time of the DNA. Both of these phenomena were reversed by addition of exogenous putrescine.

Stability and structure of model DNA triplexes and quadruplexes and their interactions with small ligands.

This review focuses on the structural and thermodynamic characterization of model DNA triplex and quadruplex structures, taking into account effects of stoichiometry and sequence. Methods such as gel electrophoresis, UV melting, and scanning calorimetry, and the results thereof, are described for determination of the thermodynamic stability of such systems. Three classes of triplexes are considered based on the composition of the third strand, while quadruplex systems are limited to those based on the guanine quartet. X-ray crystallography and high resolution NMR studies are also described for these two classes of unusual structures. Ligand binding to triplexes and quadruplexes is also reviewed, with emphasis on specific molecular recognition. The availability of three-dimensional structures for triplex and quadruplex species sets the stage for structure-based development of ligands capable of binding to them specifically. To this end, we consider the application of DOCK, a program for the discovery of small molecules that can recognize macromolecular structures, to the problem of recognizing folded quadruplex structures. Such studies may ultimately lead to pharmaceutically active compounds.

DNA damage in rat 9L cells treated with nitrogen mustard and 1,3-bis(2-chloroethyl)-1-nitrosourea assayed by viscoelastometry and S1 nuclease.

The techniques of viscoelastometry and S1 nuclease digestion were applied to the analysis of DNA damage in rat 9L cells treated with nitrogen mustard and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Results of the S1 nuclease assay permitted quantitation of the amount of single-strand (or alkali-labile) break formation as well as DNA interstrand cross-link formation. In the presence of 2% detergent, only cells treated with nitrogen mustard showed evidence of DNA cross-link formation as determined by this assay. Viscoelastic analysis of cell lysates under denaturing conditions (pH 12.15) showed that cells treated with nitrogen mustard led to substantial increases in both the viscoelastic retardation time and recoil, consistent with the presence of DNA cross-links, while treatment with BCNU led to decreases in these two properties, consistent with the induction of single-strand breaks. Viscoelastic analysis of cell lysates under nondenaturing conditions (pH 11.15) showed that nitrogen mustard produced an increase in retardation time, consistent with single-strand break induction, along with a fast recoiling component that eventually led to gel-like behavior, suggesting the possibility of drug-induced intermolecular DNA-DNA cross-links. BCNU treatment resulted in a decrease in retardation time. This decrease in consistent with induction of DNA interstrand cross-links by BCNU and shows that the single-strand breaks observed at denaturing conditions were due to the presence of alkali-labile sites rather than true strand breaks. While other methods using denaturing conditions have resulted in evidence for DNA cross-links following BCNU treatment, both viscoelastic and S1 nuclease experiments showed negative results in this regard. Further work is needed to clarify this point.

Noninvasive spectroscopic analysis of fluoropyrimidine metabolism in cultured tumor cells.

Nuclear magnetic resonance spectroscopy is a technique that may be used noninvasively to follow the intracellular metabolism of fluorinated antimetabolites such as 5-fluorouracil (FUra) and 5-fluorouridine. Intracellular 19F spectral peaks are assigned by comparison with the pH-dependent chemical shifts measured for eight commercially available fluoropyrimidine metabolites as well as by comparison with the literature recorded values of five known catabolites of FUra. Five murine and human tumor cell lines (N1S1, Sarcoma 180, L1210, HL-60, and Mia-PaCa) were exposed in vitro for 24 h to cytostatic doses of FUra or 5-fluorouridine. Treated cells were harvested and analyzed immediately or following a subsequent incubation under either nutrient-rich or nutrient-poor conditions. A major narrow component peak at 4.6-4.9 ppm was observed in all cell samples analyzed immediately after treatment. This peak was identified as intracellular FUra nucleotides, and its T1 value was approximately 800 ms. No fluoropyrimidine catabolites were detectable in any of the treated cell lines. Free FUra could be measured in cells only after subsequent incubation under nutrient-poor conditions, and this was associated with a decline in the prominent FUra nucleotide peak. In treated cells chased with drug-free media containing 1 microM thymidine, spectra revealed a broad component signal underlying and downfield from the narrow nucleotide-containing peak. By biochemically fractionating treated cells into an acid-soluble fraction and phenol-purified cytoplasmic and nuclear RNA extracts, we were able to completely separate the nucleotide peak from the broad component signal resulting from FUra incorporation into RNA. Thymidine produced a marked enhancement of this 19F signal into both cytoplasmic and nuclear RNA without affecting the nucleotide signal from the acid-soluble fraction. The present ability of nuclear magnetic resonance to monitor the metabolic channeling of fluoropyrimidines in intact tumor cells suggests that future spectroscopic imaging of patients treated with fluorinated antimetabolites may provide clinically important information about tumor biochemistry and drug sensitivity.

In vivo 19F-NMR of 5-fluorouracil incorporation into RNA and metabolites in Escherichia coli cells.

19F resonances from RNA with 5-fluorouracil incorporated could be observed in intact Escherichia coli cells, as well as in tRNA isolated from the cells. 19F-NMR signals from the metabolic breakdown products of the fluorinated RNA were also detected in vivo. By observing the 19F-NMR spectrum, variations in the metabolic disposition of administered 5-fluorouracil could be monitored as a function of time and be compared when the cells were deprived of oxygen and other nutrients, subjected to ethidium bromide treatment, or grown in the presence of mitomycin C.

Lead is unusually effective in sequence-specific folding of DNA.

DNA quadruplex structures based on the guanine quartet are typically stabilized by monovalent cations such as K(+), Na(+), or NH(+)(3). Certain divalent cations can also induce quadruplex formation, such as Sr(2+). Here we show that Pb(2+) binds with unusually high affinity to the thrombin binding aptamer, d(GGTTGGTGTGGTTGG), inducing a unimolecular folded structure. At micromolar concentrations the binding is stoichiometric, and a single lead cation suffices to fold the aptamer. The lead-induced changes in UV and CD spectra are characteristic of folded quadruplexes, although the long wavelength CD maximum occurs at 312 nm rather than the typical value of 293 nm. The one-dimensional exchangeable proton NMR spectrum shows resonances expected for imino protons involved in guanine quartet base-pairing. Furthermore, two-dimensional NMR experiments reveal NOE contacts typically seen in folded structures formed by guanine quartets, such as the K(+) form of the thrombin aptamer. Only sequences capable of forming guanine quartets appear to bind Pb(+2) tightly and change conformation. This sequence-specific, tight DNA binding may be relevant to possible genotoxic effects of lead in the environment.

1H and 31P nuclear magnetic resonance studies of spermine binding to the Z-DNA form of d(m5CGm5CGm5CG)2. Evidence for decreased spermine mobility.

The binding of spermine to the d(m5CGm5CGm5CG) duplex has been studied by proton and phosphorus nuclear magnetic resonance techniques in order to investigate the mobility and nature of spermine bound to the resulting Z-DNA complex. A characterization of the B to Z transition as a function of increasing spermine concentration demonstrated doubling of the non-exchangeable proton and the phosphorus peaks at a ratio of about 1:1 (spermine/duplex) and a re-simplification of the spectrum at 2:1 (spermine/duplex) where about 90% or the DNA was fully converted into the Z-form. However, some of the Z-DNA proton chemical shifts differed between the 1:1 and 2:1 titration points. Since these differences involved primarily the exchangeable terminal imino and amino protons, they could result from end effects. Discrepancies were generally not observed with the non-terminal proton shifts nor with the phosphorus shifts. These proton and phosphorus chemical shift changes are fully consistent with a B to Z transition. Complexed spermine peaks appear about 0.1 parts per million upfield from the uncomplexed form. The spermine and both the B and Z-DNA hexamer signals are noticeably broadened at the 1:1 ratio but the remaining signals re-sharpen at the 2:1 ratio. Both one-dimensional and two-dimensional studies revealed negative nuclear Overhauser effect (NOE) contacts between each spermine proton. Therefore, spermine has a longer correlation time than that observed for unbounded spermine. These results are contrasted with the positive NOE contacts observed for the B-DNA-spermine complexes reported by Wemmer et al. using the dodecamer d(CGCGAATTCGCG)2 and reported here using the hexamer d(ATGCAT)2. While the mobility of spermine in the Z-DNA complex is significantly less than that of the B-DNA complex, no clear evidence of intermolecular spermine-DNA proton NOE contacts is observed.

Computer modeling of actinomycin D interactions with double-helical DNA.

We have performed molecular mechanical calculations on intercalation complexes of actinomycin D with a series of base-paired hexanucleoside pentaphosphates; d(GCGCGC)2, d(GCCGGC)2, d(GCATGC)2, d(GCTAGC)2 and d(ATGCAT)2. Our results are in good agreement with previous experimental work on sequence selectivity. The results provide a rationalization for the strong preference of actinomycin D to intercalate on the 3' side of guanine residues, consistent with previously proposed models. Finally, the computed structures for d(ATGCAT)2-actinomycin D complexes have been compared with two-dimensional nuclear magnetic resonance nuclear Overhauser effect experimental results. To our knowledge, this is the first extensive comparison of molecular mechanical model structures for a drug-DNA complex with experimental solution phase data. We find generally good agreement between our computational models and the experimental solution phase structures.

NMR investigation of the interaction of mithramycin A with d(ACCCGGGT)2.

The binding of mithramycin A to d(ACCCGGGT)2 has been investigated by one- and two-dimensional 1H NMR spectroscopy. Titration of the drug into the octamer solution results in loss of the oligonucleotide C2 symmetry at stoichiometric ratios less than 4 drug molecules per duplex. However, at a ratio of 4:1 (drug/duplex), the C2 symmetry of the oligonucleotide is restored. From these data it is evident that more than one complex forms at ratios less than 4:1 while only one complex predominates at the ratio 4:1. This is the first report of a DNA octamer which binds 4 large drug molecules. These results are compared to those we have recently reported for mithramycin binding to d(ATGCAT)2, where only a single, bound complex is observed, with a stoichiometry of 2:1.

Mono-, di- and trimeric binding of a bis-netropsin to DNA.

An unusual 3:1 stoichiometry for complex formation between an elongated bis-netropsin compound and its binding site on DNA has been observed. Circular dichroism measurements distinguish two types of complexes formed between this bis-netropsin and poly[d(A-T)].poly[d(A-T)]. The first type is characterized by a 1:1 saturating ratio of bound molecules per ten base pairs. Formation of the second type results from the cooperative binding of two additional bis-netropsin molecules to the first type of complex. In contrast to these results observed for binding to the alternating polynucleotide, only the 1:1 type of complex is formed when this ligand binds to the homopolymer poly(dA).poly(dT).

NMR studies of drug-DNA complexes.

The application of high-resolution, multidimensional NMR techniques to the problem of determining the structure of drug-DNA complexes in solution has led to substantial progress in understanding the effect of drugs on DNA at the molecular level. With the development of isotopic labeling methods applied in three- and four-dimensional experiments, we anticipate that more complex drug-DNA systems will become amenable to structural analysis. In addition to implementing these newer techniques, progress will also be made in terms of investigating the structure of drug complexes with more unusual forms of DNA, such as triplexes, quadruplexes, multistranded junctions, and so forth.

Filter elution analysis of the effect of alpha-difluoromethylornithine on X-ray-induced DNA damage in 9L cells.

We used the filter elution technique to study DNA single- and double-strand scission under denaturing alkaline and nondenaturing conditions in X-irradiated 9L rat brain tumor cells. The amount of DNA damage determined by the alkaline elution assay was similar for different lysis conditions (sodium dodecyl sulfate and sarkosyl) and DNA fluorometric assays (Hoechst 33258 and 3,5-diaminobenzoic acid dyes). Therefore, results of the filter elution assay obtained with the various methods can be compared directly. Using these assays, we found that there was no significant change in the susceptibility to X-ray-induced DNA damage, measured either as single- or double-strand breaks, in 9L cells depleted of polyamines by treatment with alpha-difluoromethylornithine. Results obtained by filter elution are different from results obtained with viscoelastometry, which suggests that the two methods may resolve the effects of changes in DNA structure in different ways.

Radial migration of DNA molecules in cylindrical flow. III. Circles and the effect of non-gaussian polymer statistics.

We have previously shown that DNA will migrate radially inward in a concentric-cylinder shear flow apparatus. We assumed gaussian chain statistics, and we considered only linear molecules. In this paper, we extend the analysis to closed circular molecules, and we consider non-gaussian statistics for both linears and circles. We find that, in good solvents, the inward radial migration velocity is more sensitive to the molecular weight than M5/2, which we previously reported for gaussian chains. Furthermore, linears migrate radially inward 8 times faster than do circles of the same molecular weight. This suggests the possibility of separating linear from circular DNA in solution.

The conformation of the d(ACCCGGGT) duplex in aqueous solution.

The nonexchangeable base and sugar protons of the octanucleotide d(ACCCGGGT)2 have been assigned using two dimensional homonuclear Hartmann-Hahn relayed spectroscopy (HOHAHA), double quantum filtered homonuclear correlation spectroscopy (DQFCOSY) and nuclear Overhauser spectroscopy (NOESY) in D2O at 12 degrees C. The observed NOE's between the base protons and their own H2' protons and between the base protons and the H2' protons of the 5' adjacent nucleotide and the observed coupling constants between the deoxyribose 1' and 2',2'' protons indicate that this duplex assumes a right-handed B-type helix conformation in solution.

Tetraplex structure formation in the thrombin-binding DNA aptamer by metal cations measured by vibrational spectroscopy.

Formation of intramolecular tetraplex structures by the thrombin-binding DNA aptamer (TBA) in the presence of K(+), Pb(2+), Ba(2+), Sr(2+) and Mn(2+) has been studied by vibrational spectroscopy. All tetraplex structures contain G-G Hoogsteen type base pairing, both C2'endo/anti and C2'endo/syn deoxyguanosine glycosidic conformations and local B like form DNA phosphate geometries. Addition of Pb(2+) ions modifies the structure by interacting at the level of the guanine carbonyl groups. The very important downshift of the guanine C6=O6 carbonyl vibration mode in the TBA spectrum induced by the addition of one Pb(2+) ion per TBA molecule is in agreement with a localization of the metal ion between both guanine quartets. FTIR melting experiments show an important stabilization of the tetraplex structure upon addition of Pb(2+) ions (DeltaT = 15 degrees C). This strong interaction of lead cations may be correlated with a change in the geometry of the cage formed by the two guanine quartets. A similar but weaker effect is observed for barium and strontium cations.

Recognition of Z-RNA and Z-DNA determinants by polyamines in solution: experimental and theoretical studies.

Protonated polyamines are among the most efficient cations that induce the left-handed Z-form in certain polynucleotides. It is not known, however, whether these cations bind to specific sites on Z-sequences in solution. We have studied potential polyamine binding sites by measuring the effects of polyamines on the binding of purified immunoglobulins (IgGs) to different regions of the Z-helix and by molecular mechanics modeling. The specific binding of anti-Z-DNA and anti-Z-RNA IgGs to Z-helices was studied as a function of spermidine or spermine concentration. The effect of polyamines on the antibody-nucleic acid interaction was different for IgGs with different specificities for various determinants on the Z-helix. Polyamines inhibit the binding of certain anti-Z IgGs directed against specific sites probably at or near the interface between the major convex surface and the phosphate backbone, most likely by competing with the antibody binding site(s). In contrast, polyamines have no effect on other anti-Z IgGs directed against sites determined by the phosphate backbone. Furthermore, these cations can enhance the binding of anti-Z IgG directed against bulky groups at the C-5 position on the major convex surface of the helix; the enhancement may be related to charge neutralization. Under these conditions, no direct binding of antibodies with polyamines was observed. These data suggest the existence of a specific binding site(s) for polyamines on both Z-DNA and Z-RNA in solution. These binding sites have some similarity to those observed in oligonucleotide crystals by Quigley (in "Molecular Structure and Biological Activity," J.F. Griffin and W.L. Duax, eds., Elsevier, Amsterdam (1982), pp. 317-331). The experimental evidence for specific spermine binding sites on the helical surface was supported by molecular mechanics modeling of the interaction of spermine with the major groove of (dG-dC)5.(dG-dC)5 in both the Z- and B-forms. The crystal coordinates of spermine-containing oligonucleotides in both the B- and Z-forms were used as the starting points for modeling studies. The potential energy of spermine bound to the major convex surface of the Z-form was much less favorable than that of spermine bound to the major groove of the B-form. In the presence of sodium ions, however, the Z-form-spermine complexes were favored over the B-form. Thus, both theoretical and experimental studies indicate that polyamines can specifically recognize Z-helical determinants in solution as well as in crystals.

Design of sequence-specific DNA binding ligands that use a two-stranded peptide motif for DNA sequence recognition.

The design and DNA binding activity of beta-structure-forming peptides and netropsin-peptide conjugates are reported. It is found that a pair of peptides-S,S'-bis(Lys-Gly-Val-Cys-Val-NH-NH-Dns)-bridged by an S-S bond binds at least 10 times more strongly to poly(dG).poly(dC) than to poly(dA).poly(dT). This peptide can also discriminate between 5'-GpG-3' and 5'-GpC-3' steps in the DNA minor groove. Based on these observations, new synthetic ligands, bis-netropsins, were constructed in which two netropsin-like fragments were attached by means of short linkers to a pair of peptides-Gly-Cys-Gly- or Val-Cys-Val-bridged by S-S bonds. These compounds possess a composite binding specificity: the peptide chains recognize 5'-GpG-3' steps on DNA, whereas the netropsin-like fragments bind preferentially to runs of 4 AT base pairs. Our data indicate that combining the AT-base-pair specific properties of the netropsin-type structure with the 5'-GpG-3'-specific properties of certain oligopeptides offers a new approach to the synthesis of ligands capable of recognizing mixed sequences of AT- and GC-base pairs in the DNA minor groove. These compounds are potential models for DNA-binding domains in proteins which specifically recognize base pair sequences in the minor groove of DNA.