Ethidium bromide intercalation and chromatin structure: A thermal analysis

Abstract

Differential Scanning Calorimetry has been performed in the temperature range 310 K-410 K on intact thymocytes and physiologically isolated chromatin following Ethidium bromide intercalation. Native thymocytes exhibited four main thermal transitions (at 339 K, 347 K, 362 K and 375 K) that were assigned to the melting of different cellular components. At increasing dye concentrations an enthalpy redistribution became evident between the thermal transition at 362 K related to the melting of nucleosome organized in the 10 nm filament, and the transition at 375 K related to the melting of nucleosome organized in the 30 nm (or more) fiber. In correlation with increasing concentrations of Ethidium bromide, the disappearance and the subsequent reappearance of the highest temperature transition seem to be related to the unwrapping and subsequent wrapping of the chromatin fiber. Under similar condition, free DNA and digested chromatin do not show any enthalpy redistribution in their calorimetric profiles following Ethidium bromide intercalation. On the contrary, physiologically isolated chromatin displayed similar enthalpy redistribution between transitions assigned to chromatin DNA melting. An interesting difference appeared in the calorimetric profile of isolated chromatin with respect to the in situ material after chromatin extraction. In fact, a transition at 354 K, probably related to the melting of linker DNA became apparent (the transition at 362 K was assigned to the melting of DNA around the core particle). Selective digestions with different enzymes (micrococcal nuclease, proteinase K and DNase I) were carried out on thymocytes to verify the assignment of the main thermal transitions. In order to clarify the nature of the high temperature transitions native thymocytes were treated with topoisomerase I that removes superhelical turns from topologically closed DNA molecules. A comparison of calorimetric data with thermal denaturation profiles obtained by spectropolarimetric measurements on physiologically isolated chromatin gave further confirmation to the peak assignment by distinguishing the thermal transitions related to protein denaturation from the ones assigned to chromatin-DNA.