Effects of Hyperthermia on the Sedimentation of Nucleoids from HeLa Cells in Sucrose Gradients

Abstract

ROTI ROTI, J. L., AND PAINTER, R. B. Effects of Hyperthermia on the Sedimentation of Nucleoids from HeLa Cells in Sucrose Gradients. Radiat. Res. 89, 166-175 (1982). The changes in sedimentation distance of nucleoids in neutral sucrose gradients in the presence of various concentrations of the intercalating dye ethidium bromide are believed to reflect changes in the amount of DNA supercoiling within the nucleoid. When HeLa cells were heated to 450C for 30 min before cell lysis, sedimentation of their nucleoids was markedly affected; at all concentrations of ethidium bromide in the gradient, nucleoids from heated cells sedimented farther than those from unheated cells. After cellular protein was labeled with [3H]leucine for 15 to 17 hr, approximately 4% of the total 3H radioactivity cosedimented with nucleoids from unheated cells. This percentage increased with heating time (10-70 min) and temperature (44-460C); e.g., after 20 min at 450C the fraction of 3H cosedimenting with nucleoids was 7%. The heat-induced change in nucleoid sedimentation distance was a linear function of the percentage of 3H cosedimenting with nucleoids. These results show that hyperthermia causes an increase in the protein content of nucleoids. The additional protein affects sedimentation by increasing the mass of the nucleoids and by decreasing the amount of DNA available for supercoiling and thereby the efficiency of DNA rewinding.