ETHIDIUM-BROMIDE INTERCALATION AND CHROMATIN STRUCTURE - A THERMAL-ANALYSIS

Differential Scanning Calorimetry has been performed in the temperatur e range 310 K - 410 K on intact thymocytes and physiologically isolate d chromatin following Ethidium bromide intercalation. Native thymocyte s exhibited four main thermal transitions (at 339 K, 347 K, 362 K and 375 K) that were assigned to the melting of different cellular compone nts. At increasing dye concentrations an enthalpy redistribution becam e evident between the thermal transition at 362 K related to the melti ng of nucleosome organized in the 10 nm filament, and the transition a t 375 K related to the melting of nucleosome organized in the 30 nm (o r more) fiber. In correlation with increasing concentrations of Ethidi um bromide, the disappearance and the subsequent reappearance of the h ighest 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 redistributio n in their calorimetric profiles following Ethidium bromide intercalat ion. On the contrary, physiologically isolated chromatin displayed sim ilar enthalpy redistribution between transitions assigned to chromatin DNA melting. An interesting difference appeared in the calorimetric p rofile of isolated chromatin with respect to the in situ material afte r chromatin extraction. In fact, a transition at 354 K, probably relat ed to the melting of linker DNA became apparent (the transition at 362 K was assigned to the melting of DNA around the core particle). Selec tive digestions with different enzymes (micrococcal nuclease, proteina se K and DNase I) were carried out on thymocytes to verify the assignm ent 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 clo sed DNA molecules. A comparison of calorimetric data with thermal dena turation profiles obtained by spectropolarimetric measurements on phys iologically isolated chromatin gave further confirmation to the peak a ssignment by distinguishing the thermal transitions related to protein denaturation from the ones assigned to chromatin-DNA. (C) 1997 Elsevi er Science B.V.