Fluorescence anisotropy decay of ethidium bound to nucleosome core particles. 2. The torsional motion of the DNA is highly constrained and sensitive to pH

The effects of pH on the torsional flexibility of DNA bound to nucleosome core particles were investigated by using time-resolved fluorescence anisotropy decays of intercalated ethidium. The decays were collected by using time-resolved single-photon counting and were fit to a model developed by J. M. Schurr [(1984) Chem. Phys. 84, 71-96] with a nonlinear least-squares-fitting algorithm developed for this purpose. As the torsional flexibility of DNA is affected by the presence of an intercalating dye, the decays were studied at different ethidium bromide to core particle binding ratios. Because we see large increases in DNA flexibility and in the rotational diffusion coefficient at binding ratios of 0.6 ethidium/core particle and above, we conclude that, under these conditions, the DNA begins to detach from the protein. At lower binding ratios, we observe only small changes in the anisotropy decay. The torsional parameters obtained are a function of N, the number of base pairs of DNA between points of attachment to the histone core. Only if N is greater than 30 base pairs is the torsional rigidity of DNA on a nucleosome core particle higher than that for DNA free in solution. Also, for reasonable values of N (less than 30), the friction felt by the DNA on a core particle is much higher than that felt by free DNA. This indicates that the region of the DNA to which the ethidium binds is highly constrained in its motions. pH changes nearly neutrality at moderate ionic strengths (100 mM) have a substantial effect on the fluorescence anisotropy decays, particularly at early times. These analyses indicated that the observed change on increasing pH can be attributed either to a loosening of the contacts between the DNA and the histone core (increasing N) or to a substantial relaxing of the torsional rigidity of the DNA.     

Cellular pharmacology of cis and trans pairs of platinum complexes in cisplatin-sensitive and -resistant human ovarian carcinoma cells

The cellular pharmacology of two pairs of cis and trans platinum complexes has been studied in three human ovarian carcinoma cell lines, a parental relatively cisplatin-sensitive line (CH1), a subline possessing acquired cisplatin resistance (3-fold; CH1cisR) and an intrinsically cisplatin resistant line (13-fold; SKOV-3). Growth inhibition studies showed that both JM335 [trans ammine (cyclohexylaminedichloro dihydroxo) platinum(IV)] and its platinum(II) dichloro homolog JM334 were relatively less cross-resistant against both acquired and intrinsic cisplatin resistant cells. In contrast, resistance circumvention was not apparent in these cell lines with their cis isomeric counterparts (JM149 for JM335 and JM118 for JM334). The trans compound JM335 was more potent than its cis isomer against all three cell lines. There was no clear correlation between intracellular accumulation following 2 h exposure to each compound and resulting DNA platination or growth inhibition. The selective activity of the trans platinum complexes against the SKOV-3 cell line correlated with a deficiency in the repair of adducts within a fragment of the N-ras gene induced by trans compounds whereas adducts induced by the cis counterparts, and cisplatin, were repaired. The CH 1 parental line appeared repair deficient at the gene-specific level to adducts induced by both cis (including cisplatin) and trans compounds. Resistance in CH1cisR was associated with a lack of gene-specific repair of lesions formed by JM118 and JM149. All four compounds induced apoptosis in all three cell lines, as measured by fluorescent microscopy and field inverted gel electrophoresis, although the kinetics of apoptosis was markedly faster for the trans versus cis compounds. In summary, the trans platinum complexes JM335 and JM334 possess unique cellular properties compared to their cis counterparts particularly with respect to gene specific repair of DNA adducts and the rate of induction of apoptosis.     

Fluorescence anisotropy of DNA/DAPI complex: torsional dynamics and geometry of the complex.

Fluorescence depolarization of synthetic polydeoxynucleotide/4'-6-diamidino-2-phenylindole dihydrochloride complexes has been investigated as a function of dye/polymer coverage. At low coverage, fluorescence depolarization is due to local torsional motions of the DNA segment where the dye resides. At relatively high coverage, fluorescence depolarization is dominated by energy transfer to other dye molecules along the DNA. The extent of the observed depolarization due to torsional motion depends on the angle the dye molecule forms with the DNA helical axis. A large torsional motion and a small angle produce the same depolarization as a small torsional motion and a large projection angle. Furthermore, the extent of transfer critically depends on the relative orientation of dye molecules along the DNA. The effect of multiple transfer is examined using a Monte Carlo approach. The measurement of depolarization with transfer, at high coverage, allows determination of the dye orientation about the DNA helical axis. The value of the torsional spring constant is then determined, at very low coverage, for few selected polydeoxynucleotides.     

Investigation of DNA dynamics and drug-DNA interaction by steady state fluorescence anisotropy.

We have used steady-state fluorescence polarization anisotropy (FPA) of ethidium probe molecules bound to DNA to investigate DNA-DNA interactions and the effect of high densities of intercalating drugs on the internal motions of DNA responsible for depolarization of the ethidium fluorescence. To calibrate the method, we examined the effect of DNA length on (FPA) using DNA varying in size from 10-150 base pair. The association of approximately 30 base pair DNA at high concentrations was then detected by its effect on (FPA). With sample concentrations approaching those commonly used in various physical experiments (NMR, Raman) significant DNA-DNA interactions are observed. With high molecular weight DNA (greater than 500 base pair), the limiting value of the (FPA) (0.23) is due to internal motions of the DNA (and bound chromophores). The (FPA) of ethidium probe molecules (1 drug/200 base pair) is unaffected by the addition of high levels (1 drug/2 base pair) proflavine. This indicates that either the elastic properties of DNA are unaffected by high densities of intercalated drug or that the depolarization of the ethidium fluorescence is due to highly localized motions of the base pairs that are unperturbed by binding of drugs at neighboring sites.     

Dynamics of Z-form DNA

The torsional and bending rigidities of Z-form DNA have been studied by nanosecond fluorescence anisotropy measurements of intercalated ethidium. The results suggested that Z-form DNA was considerably more flexible than B-form DNA. We have investigated the temperature dependence of the rigidity of B- and Z-form DNA and found that the temperature dependence of the torsional rigidity of Z-form DNA was remarkably lower than that of B-form DNA.     

Effects of monofunctional platinum binding on the thermal stability and conformation of a self-complementary 22-mer

We investigated the effect of various monofunctional platinum complexes on the thermal stability and conformation of a self-complementary 22-mer duplex oligonucleotide by means of CD and UV melting profiles. We studied several families of triamine complexes of the general formula PtA2AmCl where A2=(NH3)2 and ethylenediamine and where Am=N1-4-methyl-pyridine, N7-guanosine, and 9-ethyl-guanine. Platination by the N1-4-methyl-pyridine and 9-ethyl-guanine complexes led to a decrease in the Tm of the oligonucleotide by 2-11.5 degrees C while platination with the N7-guanosine complexes led to a rise in the melting temperature of the oligonucleotides by 4.5 degrees C. A similar inverse correlation between the two groups of platinum compounds was found in the CD spectra. In all cases, the cis isomer had a more pronounced effect on both the melting curve and the CD spectrum. The cis isomer was found to have a more destabilizing effect than its trans counterpart. This indicates that the cis geometry in fact forces a greater structural constraint on the backbone of the double helix. We have also found that the sugar of the guanosine has a significant influence on both the Tm and CD spectra; the sugar moiety contributes to the stability of the double helix, probably through the formation of hydrogen bonds.     

Renaturation effects of cis and trans platinum II and IV compounds on calf thymus deoxyribonucleic acid.

The effects of cis dichlorodiammine platinum [cis Pt(II)], trans dichlorodiammine platinum (trans Pt(II)], cis tetrachlorodiammine platinum [cis Pt(IV)], trans tetrachlorodiammine platinum [trans Pt(IV)], and ethylenediaminedichloride platinum [Pt(II)en] on the absorption spectra, and thermal hyper- and hypochromicity of calf thymus DNA were investigated. Platinum-induced renaturation was studied as one parameter of interstrand cross-linking. Based on a DNA cross-linking hypothesis, the tumor-inhibitory platinum compounds cis Pt(II), cis Pt(IV) and Pt(II)en would be expected to induce renaturation following thermal denaturation, whereas the ineffective drugs, trans Pt(II) and trans Pt(IV) would not. All five bind to DNA in such a way as to induce renaturation. However, cis Pt(IV) requires at least a 3- to 4-fold longer incubation time than is required by the other compounds to form the coordination bonds necessary for renaturation. Maximum renaturation with all compounds was observed at a molar Pt/base ratio of 0.05 except cis Pt(IV), with which it was 0.25. The rate of the formation of the platinum-coordinated cross-links by fresh cis Pt(II) suggests two reactions or types of reactions occur. The first is rapid and destabilizes the DNA helix, whereas the second is slow and responsible for renaturation following thermal denaturation. These results suggest that cis Pt(IV) may be activated cellularly and that cross-linking is not the primary mechanism of action of the tumor-inhibitory platinum compounds.     

Anisotropic overall and internal motions of short DNA fragments.

Anisotropic motions of DNA fragments in the size range 6-118 base pairs are studied by the steady-state fluorescence polarization of different excitation transitions in the intercalated ethidium cation. Calculated effective tumbling and twisting times are found to be shorter than predicted for overall motions of rigid DNA, indicating that internal motions and/or dye wobbling contribute to the depolarization. The data are consistent with a model where the DNA fragments are considered to be rigid against bending but torsionally flexible, and where the dye can wobble within the intercalated site. We also discuss the possibility of correlated out-of-plane motions of the dye and the DNA bases.     

DNA adducts of antitumor trans-[PtCl2 (E-imino ether)2].

It has been shown recently that some analogues of clinically ineffective trans-diamminedichloroplatinum (II) (transplatin) exhibit antitumor activity. This finding has inverted the empirical structure-antitumor activity relationships delineated for platinum(II) complexes, according to which only the cis geometry of leaving ligands in the bifunctional platinum complexes is therapeutically active. As a result, interactions of trans platinum compounds with DNA, which is the main pharmacological target of platinum anticancer drugs, are of great interest. The present paper describes the DNA binding of antitumor trans-[PtCl(2)(E-imino ether)(2)] complex (trans-EE) in a cell-free medium, which has been investigated using three experimental approaches. They involve thiourea as a probe of monofunctional DNA adducts of platinum (II) complexes with two leaving ligands in the trans configuration, ethidium bromide as a probe for distinguishing between monofunctional and bifunctional DNA adducts of platinum complexes and HPLC analysis of the platinated DNA enzymatically digested to nucleosides. The results show that bifunctional trans-EE preferentially forms monofunctional adducts at guanine residues in double-helical DNA even when DNA is incubated with the platinum complex for a relatively long time (48 h at 37 degrees C in 10 mM NaCIO(4). It implies that antitumor trans-EE modifies DNA in a different way than clinically ineffective transplatin, which forms prevalent amount of bifunctional DNA adducts after 48 h. This result has been interpreted to mean that the major adduct of trans-EE, occurring in DNA even after long reaction times, is a monofunctional adduct in which the reactivity of the second leaving group is markedly reduced. It has been suggested that the different properties of the adducts formed on DNA by transplatin and trans-EE are relevant to their distinct clinical efficacy.     

Internal motion of DNA in bacteriophages

We have investigated internal motion of DNA in bacteriophages by measuring fluorescence anisotropy decays of intercalated ethidium. The results showed large suppression of the internal motion of the inner DNA; the interhelix interaction of the DNA in the phage head is considered to enhance the effective viscosity of the DNA rod and to restrict the angle of the internal motion. Considering that the observed internal motion arises mainly from torsional motion of the DNA, we have calculated the movable angles of the torsional motion (the standard deviation of the torsional motion) of the DNA in the phage heads. The magnitude of the calculated movable angles indicates the extent of suppression of the DNA movement in the phage head; in lambda wild type phage, the DNA is packed most rigidly in the head and the motion is found to be restricted most severely. In a deletion mutant of lambda phage, whose inner DNA content is deficient by 17.6%, steric hindrance from the interhelix DNA interaction is decreased, and the DNA can move more easily. In T4 wild type phage, although the extent of condensation of the inner DNA is the same as that in lambda wild type phage, the DNA was fairly mobile. The presence of glucosylated hydroxymethylcytosine is suggested to influence the rigidity of the inner DNA or packaging mode of the DNA in the T4 head.     

Kinetic investigation of the DNA platination reaction: evidence for a transient adduct between deoxyribonucleic acid and cis-platinum(II)

Kinetics of the synthesis of adducts between salmon testis DNA and platinum(II) compounds were measured by their effects on DNA synthesis, circular dichroism, and ethidium bromide dependent fluorescence. Transient incorporation of [14C]cyanide into DNA adducts of of cis-diammineaquochloroplatinum(II) and respectively cis-diamminediaquoplatinum(II) compounds but not of trans-diammineaquochlorplatinum(II) was observed. A minimal kinetic scheme is derived, in which a transient monodentate DNA-platinum(II) adduct is formed in a bimolecular reaction between DNA and aquated platinum(II) compounds. Second-order rate constants are 2000-3000 M-1 min-1 for cis-diamminediaquoplatinum(II) and 280-400 M-1 min-1 for cis- and trans-diammineaquochloroplatinum(II), respectively. The dependence of pseudo-first-order rate constants is not linear for high concentrations of DNA, suggesting competitive formation of more than one primary adduct. The monodentate adducts inhibit DNA polymerase catalyzed DNA synthesis. The biomolecular reaction is followed by a rearrangement (rate constant 0.22 min-1) that gives rise to most of the decrease in the fluorescence intensity and that depends on the state of aquation of the DNA-bound platinum(II) complex. By exchange of coordinated water with a second nucleotide, the monodentate adduct can form cross-links in a reaction joining the rearrangement. Adducts containing a chloro group liberate it by hydrolysis prior to cross-linking. In the case of the trans-platinum(II) adduct, the hydrolysis is aided by the trans effect of the bound first nucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)     

A natural source of fluorescence depolarization in dye-DNA complexes

The interaction of ethidium bromide with intraphage (T₄) DNA and isolated phage (T₄) DNA has been studied. The partial polarizations of fluorescence of the dye-complexes have been measured at thermal equilibrium at various temperatures and by fast cooling to a constant lower temperature. A close comparison of the results at these two conditions and an additional analysis of them from Perrin's theorem prove that a natural source of depolarization is exhibitant in DNA-dye complexes at ordinary temperatures. This depolarization effect may be attributed to some internal motions or rotations in DNA. Alternatively, the effect may be due to conformational changes within the framework of the DNA double helix, which provide a different environment for the dye. The above depolarization effect is most effective in the temperature range 37–64°.     

The antitumor drug, cis diamminedichloro-platinum, inhibits trans plasmalemma electron transport in HeLa cells

Transplasmalemma electron transport which stimulates the growth of HeLa cells is inhibited by the antitumor drug cis diammine dichloro platinum II at concentrations which correlate with cytotoxic effects. The less cytotoxic trans diammine dichloro platinum II has very little effect on the transmembrane dehydrogenase. This selectivity contrasts with the similarity of these compounds in binding to DNA. We propose that part of the selective antitumor action of the cis compound can be based on its inhibition of the transplasmalemma dehydrogenase which stimulates growth.     

Langevin modes of macromolecules: applications to crambin and DNA hexamers

Langevin modes describe the behavior of atoms moving on a harmonic potential surface subject to viscous damping described by a classical Langevin equation. We present applications to the protein crambin and to the DNA duplex d(CpGpCpGpCpG)2 and its complex with ethidium. Our friction matrix is weighted according to surface area exposed to solvent, and results are reported for various values of the solvent viscosity and models for hydrodynamic interactions. Even for relatively small solvent friction (eta = 0.3 cp) a substantial number of modes are overdamped, and time correlation functions decay smoothly without the oscillations characteristic of gas-phase calculations. Perturbation theory starting from the gas-phase modes is accurate for many low-frequency modes (which are overdamped in the presence of solvent), but fails badly for higher modes. For correlation functions of interest to fluorescence depolarization or nmr relaxation, the plateau values are insensitive to solvent viscosity, but the relaxation times are not. The advantages and limitations of this analysis of macromolecular motions are discussed.     

Change of conformation and internal dynamics of supercoiled DNA upon binding of Escherichia coli single-strand binding protein

The influence of Escherichia coli single-strand binding (SSB) protein on the conformation and internal dynamics of pBR322 and pUC8 supercoiled DNAs has been investigated by using dynamic light scattering at 632.8 and 351.1 nm and time-resolved fluorescence polarization anisotropy of intercalated ethidium. SSB protein binds to both DNAs up to a stoichiometry that is sufficient to almost completely relax the superhelical turns. Upon saturation binding, the translational diffusion coefficients (D0) of both DNAs decrease by approximately 20%. Apparent diffusion coefficients (Dapp) obtained from dynamic light scattering display the well-known increase with K2 (K = scattering vector), leveling off toward a plateau value (Dplat) at high K2. For both DNAs, the difference Dplat - D0 increases upon relaxation of supercoils by SSB protein, which indicates a corresponding enhancement of the subunit mobilities in internal motions. Fluorescence polarization anisotropy measurements on free and complexed pBR322 DNA indicate a (predominantly) uniform torsional rigidity for the saturated DNA/SSB protein complex that is significantly reduced compared to the free DNA. These observations are all consistent with the notion that binding of SSB protein is accompanied by a gradual loss of supercoils and saturates when the superhelical twist is largely removed.     

Comparison of DNA complexation with antitumor therapeutic cis-DDP and binuclear bivalent platinum compound containing pyrazine

Conformational properties of DNA molecule upon its complexation with binuclear compounds of bivalent platinum in the cis configuration containing pyrazine ligand were studied by circular dichroism, viscometry, and dynamic birefringence. Comparison with active antitumor therapeutic cis-diamminedichloroplatinum (cis-DDP) was made. Experimental data indicates that interaction of these compounds with DNA results in the formation of coordination bond of platinum with nitrogen bases. The structure of the complex depends on the ratio of platinum and DNA concentrations in initial solution. The study of DNA protonation in complex with the binuclear coordination compound showed that the binding of platinum with DNA bases occurs at the N7 atom of guanine. It was observed a competition between the studied compound and cis-DDP for binding site on DNA. The macromolecule binds stronger to the binuclear platinum compound as compared with cis-DDP.     

Topological distributions and the torsional rigidity of DNA. A Monte Carlo study of DNA circles

Distributions of the linking number of circular DNA molecules, defined as the sum of twist and the writhing number, are obtained by Monte Carlo simulations of small, randomly closed DNA circles. We estimate the relative contributions of fluctuations in twist and writhe to the linking number distribution, as functions of DNA size. Published experimental data on topoisomer distributions in circular DNA molecules are interpreted to estimate the torsional rigidity of DNA in solution. We show that ignoring the writhe component of the linking number distribution, even for DNA circles as small as 250 base-pairs, leads to an underestimate for the torsional stiffness of the double helix. The value of the torsional modulus obtained from this analysis, C = 3.4 X 10(-19) erg cm, is from 10 to 40% larger than that estimated by others and more than twice as large as the values obtained from fluorescence depolarization or other time-resolved spectroscopic measurements. We also develop further the theoretical treatment of ring closure probabilities for DNA described in the previous article. It is shown that the torsional part of the ring closure probability, phi 0,1 (tau 0) is a periodic function of DNA length that contributes strongly to the ring closure probability for short chains but makes negligible contributions for chains over 1000 base-pairs in length.     

Prophage induction and mutagenicity of a series of anti-tumour platinum(II) and platinum(IV) co-ordination complexes

11 platinum compounds with nitrogen donor ligands, previously tested for anti-tumour activity, were studied for induction of prophage lambda and for mutagenicity in the Ames assay, with various strains of Salmonella. The compounds included cis and trans isomers of Pt(II) and Pt(IV) complexes and were tested with and without metabolic activation. All the cis compounds elicited prophage induction, whereas the trans compounds were inactive. Mutagenicity was found only in strains containing the R factor, indicating that SOS-type repair processes are required for the conversion of initial DNA lesions into mutations. Mutation induction was also influenced by the excision-repair process. The 2 trans compounds were not, or only slightly, mutagenic; all other compounds were mutagenic in at least one strain, exhibiting a 2-20-fold increase over the spontaneous background level. Addition of liver homogenate had no significant effect on the number of mutants. One compound induced exclusively frameshift mutations. The other mutagenic compounds induced frameshift mutations as well as base-pair substitutions. 7 compounds were more mutagenic for the repair-proficient than for the repair-deficient strains; only one showed the opposite effect. This suggests that for mutagenicity testing of platinum compounds, repair-proficient strains are more sensitive indicators. The differences in response of the various strains are more sensitive indicators. The differences in response of the various strains toward the compounds suggest the formation of different DNA lesions and/or a selective action of repair processes on these lesions. In general, a good qualitative correlation was observed between prophage-inducing capacity, mutagenicity in bacterial and mammalian cells and anti-tumour activity.