DNA synthesis in Balb/C-3T3 ts 2 cells is restricted by a temperature-sensitive function of late G1 phase

Abstract

ts 2 BalB/C-3T3 mouse fibroblasts are cdc mutants, which arrest late in G1, at or near the G1/S traverse, upon full expression of the heat-sensitive lesion. The kinetics of temperature inhibition of DNA synthesis in logarithmically growing cultures reveal three stages of heat inactivation. During the first generation time equivalent, normal semiconservative, semidiscontinuous replication proceeds but is reduced as cells exit and do not reenter S phase. During a second such period, a minimal rate of normal DNA synthesis is maintained. Thereafter, as the cells move into a third aborted cell division cycle, the rate of DNA synthesis increases. However, all semiconservative synthesis is then replaced by DNA repair replication. Temperature inactivation of the ts 2 protein results in shutdown of nuclear DNA synthesis. In contrast, normal replication of mitochondrial DNA proceeds at control rate throughout the first stage of temperature inactivation. Synthesis of this organellar genome is quantitatively reduced as the cells move into the second phase of heat inhibition. Titration of chromatin-bound DNA with ethidium bromide revealed that wild-type cells exhibit a changing DNA topology as the temperature is raised. Temperature-inactivated ts 2 cells behave as though their DNA has been topologically frozen in the configuration of control cells at or near entry into S phase.

Mouse Polyoma Virus and Adenovirus Replication in Mouse Cells Temperature-Sensitive in DNA Synthesis

“…The ts2 cell is therefore DNA'1/ Gi'1 [3,5,28]. These three ts gene products play no obligatory role in DNA repair repli cation [11,29], On the basis of these considerations and the data presented here it could be concluded that replication of mouse adenovirus DNA can occur in the absence of normal, semicon servative cellular DNA synthesis and does not require the participation of functional cellular DNA topoisomerase II orthe /.sCI or Is 2 gene products. This is not surprising in view of recent evidence which shows that human adenovirus DNA carries genetic in formation for specific proteins that initiate, prime and replicate the viral genome [30,31].…”