Ethidium Bromide as a Cooperative Effector of a DNA Structure

Abstract

A salt-induced cooperative conformational transition of a synthetic DNA, poly(dG-dC), is reversed by addition of ethidium bromide. Binding of the dye at high salt concentrations is highly cooperative. Circular dichroism spectra of the complex and the kinetic data support a model for this cooperative binding that is formally equivalent to the "allosteric" one proposed for oligomeric proteins by Monod et al. Thus, double-helical DNA of at least one defined sequence can undergo a cooperative conformational change in solution, with simple salts and drug molecules as antagonistic effectors. Such transitions may be involved in regulatory phenomena operating directly at the level of nucleic acid structure.The alternating synthetic polynucleotide, poly(dG-dC), changes at neutral pH in a highly cooperative way from one double-helical form to another when the salt concentration is increased above 2.5 M NaCl (1, 2). This conformational transition between the low-salt "R-form" and the high-salt "L-form" is characterized by an inversion of the circular dichroism spectrum and a redshift of the UV absorption spectrum. The transition after a shift of the salt concentration follows first-order kinetics and is rather slow, occurring at room temperature over several minutes (2).To obtain information about the molecular properties of these two double-helical structures we have studied the binding of ethidium bromide at low-and high-salt concentrations. Ethidium bromide binds to double-stranded polynucleotides, presumably by intercalation (3, 4). The drug and related molecules also have been used in fluorescence staining of chromosomes (5, 6). Striking and reproducible banding patterns have been observed. The presence of bands of varying intensity in chromosomes may be due to not only different base composition (7) but also to different conformational states of the DNA. The R-L transition of poly(dG-dC) enables us to examine the interaction of drug molecules with different double-helical structures in solution. MATERIALS AND METHODSPoly(dG-dC) was synthesized with Escherichia coli DNA polymerase I and was characterized by nearest-neighbor analysis, the melting behavior, and molecular-weight determinations (2). The molar extinction coefficient at 255 nm is 7.1 X 103. Two samples were used that had sedimentation coefficients, s2o,0,, of 1.8 S and 6.3 S in alkaline solution and 2.8 S and 6.5 S in neutral solution. These values correspond to. double-stranded structures with about 20 and 400 nucleotides per strand, respectively (2, 8). The above samples, therefore, are referred to as (dG-dC)ii and (dG-dC)ilj. Ethidium bromide was a gift from Boots Pure Drug Co. and has a molar extinction coefficient E480 nm = 5450 M-1 cm-' (9). Solutions contained 20 mM Na-phosphate (pH 6.8) and 2 mM EDTA in addition to NaCL at the indicated concentrations. (In 4.4 Binding of ethidium bromide to the nucleic acid was followed by the increase in fluorescence at 590 nm with excitation at 510 nm for nucleic-acid concentrations of 0.4-10 gM. Measurem...

Polyamine induced Z‐conformation of native calf thymus DNA

“…Increased UV absorbance around 295 nm has been found to be related to structural perturbations in the classical B-DNA helix [21]. Lafer et al [22] have earlier shown that double stranded poly(dG-dC) in 4 M NaC1 attain the Z-conformation and an observed absorbance ratio (295/260) of 0.30.…”

The Interaction of Daunomycin with Polydeoxynucleotides

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On the structure of DNA

“…Support for an SBS model appears to come from the results of studies of the optical isomerisation of the double stranded synthetic polynucleotide-poly (dG-dC) (Pohl, Jovin, Baehr and Holbrook 1972). Facile interconversion of so called Rand L forms can be achieved by a change in salt concentration alone.…”