Proflavine DNA complexes

Overlap Geometry A schematic representation of the proposed overlap geometry for proflavine intercalated into a deoxy pyrimidine(3 -5 )purine site is presented below with the (o) symbols representing the location of the phenanthridine ring protons. The mutual overlap of the two base pairs at the intercalation site involves features observed in the crystal structures of a platinum metallointercalator miniature dC-dG duplex complex (55) and the more recent proflavine miniature dC-dG duplex complex (48), as well as features derived in a linked-atom conformational calculation of the intercalation site in the proflavine DNA complex (51). [4]... 

An X-ray diffraction study of the proflavine—DNA complex gave direct evidence of intercalation, by showing one molecule of the aminoacridine stacked parallel to the base-pairs in a 1 3 ratio (Neville and Davies, 1966). Linear and circular dichroism studies of flowing solutions of all five monoaminoacridines (complexed to DNA) confirm that the acridine cations lie in planes parallel to those of the base-pairs Qackson and Mason, 1971). Finally, the free-energy of binding of aminoacridines to DNA was determined and found to be of the order... 

In proflavine-DNA complex, two principal modes of binding are known to exist, which differ in stability. One is the inside binding which is attributed to Van der Waals type interactions between a dye and the bases of DNA. The other is the outside binding which is attributed to the electrostatic interaction between a dye and a phosphate group. 

Mauss Y, Poulet P, Steibel J, et al. 1980. Interaction of proflavine-calf thymus DNA complexes with Ag+ and Hg++ ions. Studia Biophysica 81 95-96. 

The overlap of proflavine with adjacent base pairs was varied until there was approximate agreement between the experimental upfield complexation shifts (Table V) and those calculated from ring current and atomic diamagnetic anisotropy contributions from the base pairs (56). The calculated upfield shifts are somewhat smaller than the experimental complexation shifts at the proflavine protons in the synthetic DNA complex (Table V). This... 

It has been shown that DNA intercalators (ethidium bromide and proflavine) form complexes with single-stranded DNA and homopolynucleotides [3]. It was of interest, therefore, to determine the effect of intercalators on the degradation of the homopolymer (ADP-ribose)jj by the poly (ADP-ribose) glycohydrolase. 

The interaction was studied by the spectrophotometric method employed by Peacocke and Skerrett (3) to investigate the interaction between proflavine and nucleic acids and depicted by Blake and Peacocke (4) to describe the interaction of aminoacridines with nucleic acids. This method was particularly suitable for studies on 9-Amino-acridine derivatives - DNA complex because of the large metachromatic effect on the spectrum of the dyes. 

A related observation is that fully relaxed supercoiled DNA/dye complexes are somehow different from nicked circular DNA/dye complexes in the presence of the same concentration of free dye, where the binding ratios should be the same. This is readily seen in gel electrophoresis in the presence of sufficient dye concentration so that at least one, but not all, of the topoisomers is positively supercoiled. The slowest moving, presumably fully relaxed, topoisomer migrates significantly faster than the nicked circle, and this difference increases with the amount of dye present. This is not observed with chloroquine, perhaps because the effect is too small. However, it is readily apparent in the original gels of Keller0 61) in which ethidium was used to unwind the topoisomers. We have confirmed this effect for ethidium and have observed similar behavior for proflavine, 9-aminoacridine, and quinacrine. 

The steady-state FPA of large ( 500bp) calf thymus DNA/ethidium complexes is unaffected by addition of proflavine up to one per two base pairs. From this, it is concluded that the torsion constant is unaltered(168) by intercalation of proflavine. However, in our time-resolved FPA studies of linear pBR322 DNA/ethidium complexes, the torsion constant is reduced by the factor 0.60 as proflavine is added from zero to one per two base pairs.(173) Whether this discrepancy is due to a real difference between these DNAs or... 

We first describe the NMR parameters for the duplex to strand transition of the synthetic DNA poly(dA-dT) (18) with occasional reference to poly(dA-dU) (24) and poly(dA- brdU) and the corresponding synthetic RNA poly(A-U) (24). This is followed by a comparison of the NMR parameters of the synthetic DNA in the presence of 1 M Na ion and 1 M tetramethylammonium ion in an attempt to investigate the effect of counterion on the conformation and stability of DNA. We next outline structural and dynamical aspects of the complexes of poly(dA-dT) with the mutagen proflavine (25) and the anti-tumor agent daunomycin (26) which intercalate between base pairs and the peptide antibiotic netropsin (27) which binds in the groove of DNA. 

Planar dyes such as proflavine can bind to DNA by intercalation between base pairs or by stacking along the groove of the helix. The former process is favored when the nucleic acid is in excess (Nuc/D 4) and in high salt solution. We are interested in the intercalation process and hence our NMR studies have focussed on an investigation of the proflavine poly(dA-dT) complex as a function of the Nuc/D ratio in 1 M NaCl in an attempt to probe the structure and dynamics of mutagen-nucleic acid interactions in solution (25). 

Nuc/D =8, in 1 M NaCl solution with the resonance shifting to high field on complex formation (Figure 16). The results demonstrate that the base pairs are intact in the proflavine complex with the synthetic DNA. 

Melting Transition Typical 360 MHz proton NMR spectra of proflavine poly(dA-dT) complexes, Nuc/D = 24 and Nuc/D = 8, in 1 M NaCl solution at temperatures below the midpoint for the dissociation of the complex are presented in Figures 17A and B respectively. The stronger base and sugar resonances can be readily resolved from the weaker proflavine resonances (designated by asterisks) in the presence of excess nucleic acid (Figure 17) so that the resonances of the synthetic DNA and the mutagen can be monitored independently of each other. 

In the absence of DNA-histone complexes, ethacridine, proflavine, tilorone R10,556 DA, ellipticine and daunomycin inhibit the poly(ADP-ribose) glycohydrolase activity... 

Effect of DNA and DNA-Histone Complexes on the Inhibition of Poly(ADP-Ribose) Glycohydrolase by Intercalators. The inhibition of glycohydrolase activity by intercalators (proflavine, ellipticine, tilorone R10,556 DA) is relieved by the addition of 100 Mg mr calf thymus DNA (data not shown). This is probably due to the well-known binding of the intercalators to the added DNA. However, addition of DNA-histone complexes (100 jug nil" of each) slightly increases the inhibition of the glycohydrolase activity by those intercalators which are inhibitory (Table 1). These include ethacridine, tilorone R10,556 DA, eUipticine, daunomycin and proflavine. 

Fig. 1. Displacement of histone from DNA-histone complexes by intercalators 100 Mg of calf thymus DNA was added to 1 ml of 50 mM potassium phosphate buffer (pH 7.5) containing 100 Mg of histone and incubated for 15 min at 37°C. To remove the free histone, the DNA-histone complexes were washed (by centrifugation) three times with 50 mAf potassium phosphate buffer (pH 7.5), each time followed by 15 min incubation at 37°C. The washed complexes were suspended in 1 ml of 50 mM potassium buffer (pH 7.5) containing 200 mAf sodium chloride and 100 mA of the indicated intercalator. After incubation at 37° C for 30 min, the insoluble complexes were precipitated by centrifugation (8000 g 15 min, 4°C). 75 m1 of the supernatant was spotted on Whatman filter paper in 15 m1 aliquots. After drying, the filter was stained with 0.5% (w/v) Coo-massie blue in 7% (v/v) acetic acid, 30% (v/v) ethanol and destained with 7% acetic acid. 30% ethanol. 1 none, 2 proflavine, 3 ethacridine, 4 tilorone RIO,556 DA, 5 AMSA, 6 chloroquine, 7 ethidium bromide, 8-10 pure histone 0.5,1.5 and 2 Mg, respectively...

The increased inhibition of the glycohydrolase in the presence of DNA-histone could be due to the formation of dye-DNA-histone complexes or the release of inhibitory histone from DNA by the intercalators. To test this suggestion the ability of these intercalators to release histone from DNA-histone complexes was studied. The method is described in the legend to Fig. 1 the displaced histone was separated from the insoluble DNA-histone complex by centrifugation. As shown in Fig. 1, there is a correlation between the ability of the intercalators to release histone from the complexes and the increase in their inhibition of the glycohydrolase activity in the presence of DNA-histone. AMSA (spot 5) and chloroquine (spot 6), which do not inhibit the enzyme activity in the presence of DNA-histone complexes, did not release histone from these complexes. However, proflavine (spot 2), ethacridine (spot 3) and tilorone (spot 4) which are inhibitors of the glycohydrolase and also display increased inhibition of the... 

More specifically, our data following the order of exposition from relatively simple to more complex cases, mainly concern the energetics of (a) (i) the dimerization in aqueous solution of the dyes ethidium bromide (EB), proflavine (PF), and acriflavine (AF) (ii) the interaction of EB with synthetic polycarboxylates. (b) The dimerization of the antibiotics Daunomycin (D), Adriamycin (A), and Actinomycin (Act), and of the interaction of Act with deoxyguanosine-5 -phosphate (dGMP) in aqueous solution. (c) The interaction of the dyes and of the antibiotics indicated above with DNA (calf-thymus), (d) The interaction with DNA of a natural dye (and of one simple derivative) belonging to a novel class of compounds from colonial anthozoans... 

The dye, proflavine binds to double-stranded DNA tightly. When DNA is incubated with proflavine, the viscosity of the complex increases. This can happen only when the length of DNA Increases. For this, the dye must get inside the DNA structure in a manner that it lengthens the whole structure. 

Other techniques can be applied to study the same phenomenon. When DNA is incubated with proflavine, the sedimentation coefficient of the complex decreases. This can happen if DNA becomes depol5rmerized due to proflavine. It may also happen if the axial ratio of the complex increases. With the first possibility, a decrease in viscosity must take place. This is not observed. With the second possibility, viscosity should increase, and this is obserx ed. Thus, it seems that the second possibility is correct. Thus, DNA length indeed increases when it is treated with this dye. 

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