Structure of parental deoxyribonucleic acid of synchronized HeLa cells

Abstract

We have investigated the structure of parental DNA as a function of the cell cycle phase of HeLa cells. DNA was isolated from synchronized HeLa cells 0, 5, 8, and 12 h after release from a second exposure to 2 mM thymidine. These DNA preparations were characterized by CS2SO4/AgClO4 buoyant density, sensitivity to a single-strand specific nuclease, sedimentation in neutral and alkaline sucrose gradients, and sedimentation in neutral sucrose gradients after digestion with S1 nuclease. The cultures were staged according to cell cycle phase by measurements of DNA content per cell by flow microfluorometry. The cell cycle phases were G1/S (0-h culture), S (5-h culture), G2 (8-h culture), and G1 (12-h culture). There are no nuclease-sensitive sites in G2. As the cells enter G1, the number increases, with a maximum being reached in the S phase. The number of breaks in DNA with respect to cell cycle phase follows the same pattern. The amount of single strandedness, measured by buoyant density and nuclease sensitivity, is also minimal in G2, increases in G1, with a maximum achieved in the S phase. It appears that there is a chromosomal cycle, reflected as continuous structural changes in the DNA molecule, as cells traverse the cell cycle.