Restricted Hydrolysis of Newly Replicated DNA in Isolated Nuclei from Yosbida Sarcoma Cells

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Abstract
The acid solubility of radioactive nuclear DNA was examined under various conditions using isolated nuclei from [3H]thymidine-labeled Yoshida sarcoma cells. Upon incubation at 37°C, acid-soluble radioactivity increased rapidly in 30 min, and the increase continued gradually during 4 h incubation. The maximal hydrolysis was obtained at pH 7–7.5 in the presence of 0.25 mat CoCl2 MgCl2 (2 mM) replaced CoCl2 with a 30% reduced activity. The ratio of hydrolyzed to the total radioactive DNA increased in proportion to shortening of the pulse labeling times; 7% of the ratio in 8 min pulse-labeled nuclei increased to 18% in 2 min pulse-labeled and 37% in 1 min pulse-labeled nuclei. However, only 2% of the labeled DNA was hydrolyzed in nuclei isolated from cells labeled for 1 min and chased for 20 min in the presence of excess thymidine. Significant inhibition was observed upon addition of GMP, 6-mercaptopurine riboside 5′-phosphate, or deoxyribonucleoside triphosphates. The findings showed that divalent cation-dependent DNA hydrolysis was restricted to newly replicated DNA.

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