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Enzymes of Nucleic Acid Metabolism in Cells Infected with Polyoma Virus¹

Division of Biochemical Virology, Baylor University College of Medicine, Houston, Texas

The phosphorylations of thymidine-³H and deoxyuridine-³H (thymidine kinase activity) were markedly enhanced in primary cultures of murine cells 16–48 hr after polyoma virus infection. The presence of puromycin in the infected cultures from 18 to 28 hr after infection prevented this increase. If puromycin was removed at 28 hr, thymidine kinase synthesis was restored. Addition of 1-ß-D-arabinofuranosylcytosine or 5-bromodeoxyuridine to the polyoma-infected cultures at 2 hr after infection did not inhibit the increase in thymidine kinase activity observed at 28 or 48 hr. Extracts prepared from polyoma virus-infected cells did not activate the thymidine kinase of extracts from noninfected cells, nor did the latter extracts inhibit the enzyme activity of extracts from infected cells.

Thymidine kinase has been partially purified. With deoxyuridine as substrate, the Michaelis constants of the enzymes from polyoma virus-infected and noninfected cells were not significantly different. When preincubated at 38°C in the absence of substrate, the enzymes were gradually inactivated at about the same rate.

Thymidylate kinase activity was also increased in polyoma virus-infected cultures, but uridine kinase and thymidylate phosphatase activities did not increase.

Radioautographic and biochemical experiments have demonstrated that at about the time that the enzymatic increases take place, there is a significant enhancement of the capacity of virus-infected cultures to incorporate thymidine-³H into DNA.

¹ This investigation was aided by grants from the National Science Foundation (GB 3126) and the American Cancer Society (E 291), and by USPHS research grants (CA-06656, I-K6-AI-2352, and I-K3-CA-25,797).

Received 8/16/65.