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

Hiroko Yoshida, Charles E. Swenberg, Nicholas Geacintov

Research output: Contribution to journalArticle

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 φX174 DNA, have been studied by gel electrophoresis and a novel application of a kinetic flow linear dichroism technique. The magnitude of the linear dichroism (ΔA) 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 ΔA spectra of BPDE-DNA reaction mixtures vary on time scales of 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 ΔA (up to 250%, corresponding to the dichroism observed with relaxed circular DNA), followed by a slower decrease in the linear dichroism signal. This decrease in ΔA 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.

Original languageEnglish (US)
Pages (from-to)1351-1358
Number of pages8
JournalBiochemistry
Volume26
Issue number5
StatePublished - 1987

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7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide
Superhelical DNA
Ethidium
Benzo(a)pyrene
Epoxy Compounds
Conformations
Kinetics
Carcinogens
DNA Adducts
DNA
Molecules
Circular DNA
Dichroism
Intercalation
Electrophoresis
Pharmaceutical Preparations
Reaction rates
Hydrodynamics
Hydrolysis
Rate constants

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{4f424191a0e54983a9b1032811857250,
title = "Kinetic flow dichroism study of conformational changes in supercoiled DNA induced by ethidium bromide and noncovalent and covalent binding of benzo[a]pyrene diol epoxide",
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 φX174 DNA, have been studied by gel electrophoresis and a novel application of a kinetic flow linear dichroism technique. The magnitude of the linear dichroism (ΔA) 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 ΔA spectra of BPDE-DNA reaction mixtures vary on time scales of 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 ΔA (up to 250{\%}, corresponding to the dichroism observed with relaxed circular DNA), followed by a slower decrease in the linear dichroism signal. This decrease in ΔA 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.",
author = "Hiroko Yoshida and Swenberg, {Charles E.} and Nicholas Geacintov",
year = "1987",
language = "English (US)",
volume = "26",
pages = "1351--1358",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "5",

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T1 - Kinetic flow dichroism study of conformational changes in supercoiled DNA induced by ethidium bromide and noncovalent and covalent binding of benzo[a]pyrene diol epoxide

AU - Yoshida, Hiroko

AU - Swenberg, Charles E.

AU - Geacintov, Nicholas

PY - 1987

Y1 - 1987

N2 - 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 φX174 DNA, have been studied by gel electrophoresis and a novel application of a kinetic flow linear dichroism technique. The magnitude of the linear dichroism (ΔA) 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 ΔA spectra of BPDE-DNA reaction mixtures vary on time scales of 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 ΔA (up to 250%, corresponding to the dichroism observed with relaxed circular DNA), followed by a slower decrease in the linear dichroism signal. This decrease in ΔA 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.

AB - 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 φX174 DNA, have been studied by gel electrophoresis and a novel application of a kinetic flow linear dichroism technique. The magnitude of the linear dichroism (ΔA) 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 ΔA spectra of BPDE-DNA reaction mixtures vary on time scales of 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 ΔA (up to 250%, corresponding to the dichroism observed with relaxed circular DNA), followed by a slower decrease in the linear dichroism signal. This decrease in ΔA 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.

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