Spectrofluorometric Measurement of the Binding of Ethidium to Superhelical DNA from Cell Nuclei

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Abstract
Structures retaining many of the morphological features of nuclei may be released by lysing HeLa cells in solutions containing non-ionic detergents and high concentrations of salt. These nucleoids contain few chromatin proteins. The DNA of nucleoids is quasicircular and supercoiled as shown by measuring spectrofluorometrically the amount of the intercalating dye, ethidium, bound to unirradiated and .gamma.-irradiated nucleoids. Ethidium binds to nucleoids in the manner characteristic of the binding to superhelical DNA: at low concentrations more ethidium binds to unirradiated nucleoids than to their .gamma.-irradiated counterparts with broken DNA, and at higher concentrations less ethidium binds to the unirradiated nucleoids. The quasi-circles in nucleoids are 22 times less sensitive to .gamma.-irradiation than are circles of pure PM2 DNA: they must contain about 2.2 .times. 105 base pairs. The constraints that maintain the quasi-circularity of nucleoid DNA are very resistant to extremes of temperature and alkali; some remain under conditions in which the duplex is denatured. The constraints are destabilized by ethidium suggesting that they are stabilized by free energy of supercoiling. Proteolytic enzymes, but not RNases, remove the constraints. Possible structures for the constraining mechanism are discussed.