Effects of salt concentration and H1 histone removal on the differential scanning calorimetry of nuclei

• Published in: Biochemistry (Easton) Vol. 31; no. 6; pp. 1842 - 1849
• Main Authors: Touchette, Nancy A, Cole, R. David
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 18-02-1992
• Subjects: Biological and medical sciences; Calorimetry, Differential Scanning; Cell Nucleus - chemistry; Cell Nucleus - ultrastructure; Chromatin - chemistry; Chromatin - ultrastructure; DNA - chemistry; Dose-Response Relationship, Drug; Fundamental and applied biological sciences. Psychology; HeLa Cells; Histones - chemistry; Hot Temperature; Humans; Magnesium Chloride - pharmacology; Microscopy, Electron; Molecular biophysics; Nucleic Acid Denaturation; Physico-chemical properties of biomolecules; Sodium Chloride - administration & dosage; Sodium Chloride - pharmacology; Thermodynamics; Human; Differential scanning calorimetry; Chromatin; Depletion; Ionic strength; Cell nucleus; Thermal denaturation; Environmental factor; Histone H1; In vitro; Hela cell line; Nuclear protein
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00121a037

The effects of increasing NaCl concentrations on the melting profiles of chromatin in isolated nuclei contradicted published claims that structural transitions near 76 degrees C (Tn-7), near 89 degrees C (Tn-8), and near 105 degrees C (Tn-10) were respectively the melting of linker DNA, the melting of extended nucleosomal strands, and the collapse of nucleosomes in the 300-A fiber. Contrary to expectations of such an interpretation, decreases in salt concentration stabilized Tn-7 and failed to eliminate Tn-10. Moreover, nuclei depleted of H1 histone, which is known to be essential for the formation of the 300-A fiber, gave the same melting profile as intact nuclei with regard to the relative magnitudes of Tn-8 and Tn-10. The effect of salt concentration on the melting profiles and the insensitivity of Tn-8 and Tn-10 to H1 histone removal supports the notion that Tn-7 is the collapse of the nucleosome while Tn-8 and Tn-10 are respectively the unstacking of nucleotide bases in relaxed chromatin and supercoiled chromatin. The identification of Tn-8 as the unstacking of bases in relaxed DNA, and Tn-10 as unstacking in supercoiled DNA, shows that scanning calorimetry can be used to measure the state of repair of DNA in the nucleus. The gain in Tn-8 at the expense of Tn-10 that is seen as the mitotic index drops and differentiation occurs suggests that nicks accumulate in the DNA, perhaps because the gross aggregation of the inactive majority of the chromatin makes it inaccessible to repair enzymes.