Thermal denaturation of the DNA-ethidium complex. Redistribution of the intercalated dye during melting

The temperature dependence of the circular dichroism of the DNA-ethidium bromide complex at elevated temperatures provides evidence that the optical activity of the complex near 307 nm originates from interactions between intercalated dye molecules while the optical activity near 515 nm results from singly intercalated ethidium bromide molecules. The behavior of the circular dichroism of the complex at elevated temperatures also explains the higher ellipticities near 307 nm which characterize complexes formed between ethidium bromide and denaturated DNA. Finally the circular dichroism data indicate that the melting of the complex takes place in a stepwise manner with some DNA regions, probably AT-rich regions, dissociating first. The implications of these findings regarding the inhibiting effect of ethidium bromide on the function of DNA polymerase are examined.