Conversions of the left-handed form and the protonated form of DNA back tothe bound right-handed form by sanguinarine and ethidium: A comparative study

The interaction of sanguinarine and ethidium with right-handed (B-form), le ft-handed (Z-form) and left-handed protonated (designated as H-L-form) stru ctures of poly(dG-dC).poly(dG-dC) and poly(dG-me(5)dC).poly(dG-me(5)dC) was investigated by measuring the circular dichroism and UV absorption spectra l analysis. Both sanguinarine and ethidium bind strongly to the B-form DNA and convert the Z-form and the H-L-form back to the bound right-handed form . Circular dichroic data also show that the conformation at the binding sit e is right-handed, even though adjacent regions of the polymer have a left- handed conformation either in 2-form or in H-L-form. Both the rate and exte nt of B-form to Z-form transition were decreased by sanguinarine and ethidi um under ionic conditions that otherwise favour the left-handed conformatio n of the polynucleotides. The rare of decrease is faster in the case of eth idium as compared to that of sanguinarine. Scatchard analysis of the spectr ophotometric data shows that sanguinarine binds strongly to both the polynu cleotides in a non-cooperative manner under B-form conditions, in sharp con trast to the highly-cooperative binding under Z-form and H-L-form condition s. Correlation of binding isotherms with circular dichroism data indicates that the cooperative binding of sanguinarine under the Z-form and the H-L-f orm conditions is associated with a sequential conversion of the polymer fr om a left-handed to a bound right-handed conformation. Determination of bou nd alkaloid concentration by spectroscopic titration technique and the meas urement of circular dichroic spectra have enabled us to calculate the numbe r of base pairs of Z-form and H-L-form that adopt a right-handed conformati on for each bound alkaloid. Analysis reveals that 2-3 base pairs (bp) of Z- form of poly(dG-dC).poly(dG-dC) and poly(dG-me(5)dC).poly(dG-me(5)dC) switc h to the right-handed form for each bound sanguinarine, while approximately same number of base pairs switch to the bound right-handed form in complex es with HL-form of these polynucleotides. Comparative binding analysis show s that ethidium also converts approximately 2 bp of Z-form or H-L-form to b ound right-handed form under same experimental conditions. Since sanguinari ne binds preferentially to alternating GC sequences, which are capable of u ndergoing the B to Z or B to HL transition, these effects may be an importa nt part in understanding its extensive biological activities. (C) 1999 Else vier Science B.V. All rights reserved.