Effect of discrete distribution of ions on B- and Z-DNA: a theoretical investigation.
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DNA↗
Biomedical subjects
R H Shafer
Publications and source records attributed to R H Shafer.
Spectrophotometric and NMR analysis of the interaction of fluorinated mono- and bifunctional intercalators with DNA, poly(dA-dT), and poly(dG-dC).
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Acridines↗
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.
Binding Sites↗
A stopped-flow H-D exchange kinetic study of spermine-polynucleotide interactions.
The rates of H-D exchange for imino and amino protons in adenosine, calf thymus DNA, poly (dA-dT), poly(dG-dC), and poly (dG-me5dC) were determined using stopped flow kinetic methods in the presence of various concentrations of Tris, imidazole, Mg2+, and spermine in citrate buffer (pH 7, 25 degrees C). CD spectroscopic studies showed that all polynucleotides always remain in the B-form under these conditions. An increase in the concentration of Tris and imidazole from 5 mu M to 20 mM caused an increase in the rates of exchange of both fast-exchanging imino and slow-exchanging amino protons. The limiting rates of exchange at infinite concentrations of catalysts were found to be different for fast (31-57 sec-1) and slow (1-2 sec-1) exchanging protons. These results indicate that imino and amino protons of B-DNA exchange asymmetrically from two different open states as observed for Z-DNA. An increase in the concentration of spermine from a ratio of 1:50 to 1:2 of positive charge/phosphate decreased the rate of exchange of imino protons of calf-thymus DNA, poly(dG-dC), and poly(dG-me5dC), but increased the rate of exchange of the imino protons of poly(dA-dT) without affecting the exchange rate of the amino protons of any of the polynucleotides. These results are interpreted in terms of possible spermine-induced change of conformations of oligonucleotides of specific sequence that has been suggested by theoretical model building studies.
Circular Dichroism↗
NMR studies of the interaction of chromomycin A3 with small DNA duplexes I.
1H and 31P NMR spectral analysis of a chromomycin/d(ATGCAT)2 complex provides strong evidence for a nonintercalative mode of drug binding. Investigation of the imino proton region of the duplex suggests a protection of one of the two guanine imino protons from fast exchange with the bulk water up to at least 45 degrees C by the drug. Subsequent one-dimensional nuclear Overhauser enhancement experiments place the exchangeable chromomycin chromophoric hydroxyl proton less than 0.45 nm from this guanine imino proton and the chromophore 7-methyl less than 0.45 from the internal thymine 6-proton and/or the guanine 8-proton. 1H two-dimensional NMR reveals that the duplex retains a right-handed B conformation but there are distortions at the TGC region of one chain and large deviations in the chemical shift of protons relative to the uncomplexed duplex in the other chain in the same TGC region. The data suggest that the chromomycin chromophore is oriented such that the hydrophilic side of the ring system is proximal to the helix center in the major groove near the TG region while the aromatic side of the ring is oriented away from the helix but is partially protected from the solvent by the aliphatic chain, which bends back over the two aromatic protons. Changes in the 31P spectrum of the duplex on binding of the drug are different from the effect of either actinomycin or netropsin on nucleic acid fragments.
Base Composition↗
Kinetic studies of actinomycin D binding to mono-, oligo-, and polynucleotides.
The kinetics of association of actinomycin D with poly(dG-dC) and the oligonucleotides d-(CGCGCGCG), d(GCGCGC), d(ATGCAT), d(CGCG), and d(GC), as well as with several mononucleotides, have been investigated. In all cases, the association interaction was characterized by several slow, unimolecular processes with qualitatively similar rate constants. The activation enthalpies and entropies for the association of actinomycin with deoxyguanosine 5'-monophosphate resemble closely those typical for calf thymus DNA. This observation of little or no sequence or length dependence in the binding kinetics suggests that the slow phases arise from properties of the drug alone. These results are discussed in terms of both the shuffling model of Fox and Waring [Fox, K.R., & Waring, M.J. (1984) Eur. J. Biochem. 145, 579] and the model of Muller and Crothers [Muller, W., & Crothers, D.M. (1968) J. Mol. Biol. 35, 251] involving both rapidly forming and slowly forming complexes, with the rapidly forming species being the predominant one.
Base Sequence↗
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.
Magnetic Resonance Spectroscopy↗
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.
Animals↗
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.
Computers↗
Role of divalent cations on DNA polymorphism under low ionic strength conditions.
We have examined the conformational properties of poly(dG-m5dC) under a variety of low salt conditions and sample preparations. Extensive dialysis against 0.5 mM Na-cacodylate resulted in a left-handed polynucleotide conformation as determined by circular dichroism, in agreement with recently reported results. Similarly, extensive dialysis against Tris-EGTA also led to a left-handed conformation. Dilution of these samples led to a transition to the right-handed conformation. More stringent treatments such as dialysis followed by passage over an ion exchange column also resulted in a right-handed conformation. When these various solutions were examined using atomic absorption spectroscopy, significant levels of Mg+2 were observed (greater than or equal to 190 per 1000 nucleotides) in all samples showing a left-handed form, while much lower levels (less than or equal to 45 per 1000 nucleotides) were found in the low salt samples displaying a right-handed conformation. Addition of MgCl2 to samples in which divalent cations had been almost completely removed led to the reformation of the left-handed form. These results indicate that the left-handed form seen under certain low salt conditions is due to the presence of Mg+2 ions that remain bound to the polynucleotide, even in the presence of EDTA.
Cations, Divalent↗
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.
Animals↗
NMR studies of chromomycin A3 interaction with DNA.
The binding of chromomycin A3 to calf thymus DNA and poly(dG-dC) has been studied by 13C and 1H NMR with emphasis on the mode of binding, the role of Mg2+, and pH effects. The most prominent changes in the DNA base pair 13C NMR resonances upon complexation with chromomycin were observed for G and C bases, consistent with the G-C preference exhibited by this compound. Comparison of the 13C spectrum of DNA-bound chromomycin A3 with that of DNA-bound actinomycin D, a known intercalator, showed many similarities in the base pair resonances. This suggested the possibility that chromomycin A3 binds via an intercalative mechanism. 1H NMR studies in the imino proton, low-field region of the spectrum provided additional evidence in support of this binding mode. In the low-field spectrum of chromomycin A3 bound to calf thymus DNA, a small shoulder was observed on the upfield side of the G-C imino proton peak. Similarly, in the chromomycin A3 complex with poly(dG-dC), a well-resolved peak was found upfield from the G-C imino proton peak. These results are expected for ligands that bind by intercalation. Furthermore, in both the calf thymus and poly(dG-dC) drug complexes (in the presence of Mg2+) a broad peak was also present downfield (approximately 16 ppm from TSP) from the DNA imino protons. This was attributed to the C-9 phenolic hydroxyl proton on the chromomycin chromophore. Visible absorbance spectra at different pH values showed that the role of Mg2+ in the binding of chromomycin A3 to DNA is more than simple neutralization of the drug's anionic change.
Animals↗
New DNA polymorphism: evidence for a low salt, left-handed form of poly(dG-m5dC).
Spectroscopic studies on solutions of poly(dG-m5dC) over a wide range of salt concentration are presented. Low salt solutions [( Na+]) less than 2 mM) of poly(dG-m5dC) produce circular dichroism (CD) spectra typical of the left-handed, Z form at high salt [( Na+] = 1.75 M). Solutions of poly(dG-m5dC) at intermediate salt concentrations, e.g., 142 mM, yield CD spectra characteristic of the right-handed, B conformation. 31p NMR spectra of the low salt form of poly(dG-m5dC) reveal two well separated peaks, split by 1.4 ppm, consistent with a dinucleotide repeat. Kinetic studies show that the transition from the low salt form to teh right-handed B form is slow, as expected for a major conformational change. These results suggest that the Z conformation in poly(dG-m5dC) can be stabilized at very low salt as well as at high salt.
Circular Dichroism↗
Binding of ethidium and bis(methidium)spermine to Z DNA by intercalation.
The interaction of ethidium bromide, a DNA intercalating drug, and bis( methidium )spermine, a DNA bis-intercalating compound, with the left-handed Z form of poly(dG-dC) has been studied in 4.4 M NaCl. Spectrophotometric analysis using absorption, fluorescence and circular dichroism indicates that the complex formed between ethidium and Z DNA resembles very closely that formed with B DNA. This suggests that ethidium binds to Z DNA by intercalation. 31P NMR spectra are presented showing both the conversion of the Z form to the B form with increasing amounts of drug and the typical Z form spectrum at low binding densities. Data are also presented which show that the bifunctional intercalator bis( methidium )spermine binds to Z DNA in a manner similar to its binding to B DNA, i.e., by bis-intercalation. These results are important for our understanding the behavior of Z DNA and its biological significance.
Animals↗
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.
Escherichia coli↗
Aqueous solution structure of an intercalated actinomycin D-dATGCAT complex by two-dimensional and one-dimensional proton NMR.
Two-dimensional NOESY and COSY 1H NMR techniques have been employed to determine the conformation of the complex formed by actinomycin D and the hexanucleoside pentaphosphate dATGCAT. One-dimensional NOE experiments in H2O confirmed the intact nature of the oligonucleotide double helix within the complex. The drug chromophore was intercalated between the GC base pairs, with the pentapeptide lactones nestled in the minor groove. No significant conformational change of the pentapeptide lactones between bound and free drug was observed.
Dactinomycin↗
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.
Animals↗
13C-nmr study of chromomycin A3 and actinomycin D complexed with calf thymus DNA fragments.
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Animals↗