Kinetic flow dichroism study of conformational changes in supercoiled DNA induced by ethidium bromide and noncovalent and covalent binding of benzo[a]pyrenediol epoxide

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Abstract
The dynamic conformational changes due to the noncovalent intercalative binding of ethidium bromide and racemic trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), and the covalent binding of BPDE to supercoiled .vphi.X174 DNA, have been studied by gel electrophoresis and a novel application of a kinetic flow linear dichroism technique. The magnitude of the linear dichroism (.DELTA.A) of the DNA oriented in the flow gradient is sensitive to the hydrodynamic shape of the DNA molecule which is affected by the binding of the drug or the carcinogen BPDE. While the linear dichroism of ethidium bromide supercoiled DNA is time independent, the .DELTA.A spectra of BPDE-DNA reaction mixtures vary on time scales and minutes, which correspond to the reaction rate constant of BPDE to form 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene hydrolysis products and covalent DNA adducts. The rapid noncovalent intercalation of BPDE causes an initial large increase in .DELTA.A (up to 250%, corresponding to the dichroism observed with relaxer circular DNA), followed by a slower decrease in the linear dichroism signal. This decrease in .DELTA.A is attributed to the removal of intercalated diol epoxide molecules and the resulting reversible increase in the number of superhelical turns. The kinetic flow dichroism spectra indicate that the noncovalent BPDE-DNA complexes are intercalative in nature, while the covalent adducts are characterized by a very different conformation in which the long axes of the pyrenyl residues are oriented at a large angle with respect to the average orientation of the planes of the DNA bases. These results suggest that conformations of carcinogen-DNA adducts, other than intercalative ones, can cause the unwinding of superhelical DNA. The flow dichroism method is capable of following kinetically changes not only in the shapes, and thus conformations of supercoiled DNA molecules, but also in the conformations of drugs or carcinogens causing these changes.