(48 h) by procedures described previously (13,24,28). Non-temperature-sensitive revertants (ts2OR) were isolated from ts20 on the basis of spontaneous colony formation at 39°C, using cells grown from low inocula. Cell culture techniques were as previously described (13, 14). The ts mutant was main-1815
Cell and polyomavirus DNA synthesis in ts20, a temperature-sensitive mutant derived from Balb/3T3 cells, is inhibited at an early step in chain elongation in vivo and in vitro. Virus DNA synthesized under restrictive conditions, when analyzed by gel electrophoresis and fluorography, contained a series of equally spaced bands migrating between form I and form II. If restrictive conditions were prolonged, the relative amount of these less-supercoiled topoisomers increased while the overall amount of virus DNA decreased. DNA topoisomerase I activity was lower and more heat-labile when prepared from mutant cells compared to wild-type and revertant cells. An assay in which extracts from wild-type cells corrected defective cell DNA synthesis in lysed mutant cells was applied to purification of the active factor from such extracts. Salt fractionation and three cycles of column chromatography resulted in the isolation of the activity in a fraction containing 10 major polypeptides. The specific activity in the final preparation was increased fivefold and was accompanied by the activity of DNA topoisomerase I. Our results provide evidence that DNA topoisomerase I functions at an early step in chain elongation of cell and polyomavirus DNA synthesis and that the enzyme activity may be decreased as a result of the mutation in ts20.
ts20 is a temperature-sensitive mutant cell line derived from BALB/3T3 cells. DNA synthesis in the mutant decreased progressively after an initial increase during the first 3 h at the restrictive temperature. RNA and protein synthesis increased for 20 h and remained at a high level for 40 h. Cells were arrested in S phase as determined by flow microfluorimetry, and DNA chain elongation was retarded as measured by fiber autoradiography. Infection with polyomavirus did not bypass the defect in cell DNA synthesis, and the mutant did not support virus DNA replication at the restrictive temperature. After shift down to the permissive temperature, cell DNA synthesis was restored whereas virus DNA synthesis was not. Analysis of virus DNA synthesized at the restrictive temperature showed that the synthesis of form I and replicative intermediate DNA decreased concurrently and that the rate of completion of virus DNA molecules remained constant with increasing time at the restrictive temperature. These studies indicated that the mutation inhibited ongoing DNA synthesis at a step early in elongation of nascent chains. The defect in virus and cell DNA synthesis was expressed in vitro. [3H]dTTP incorporation was reduced, consistent with the in vivo data. The addition of a high-salt extract prepared from wild-type 3T3 cells preferentially stimulated the incorporation of [3H]dTTP into the DNA of mutant cells at the restrictive temperature. A similar extract prepared from mutant cells was less effective and was more heat labile as incubation of it at the restrictive temperature for 1 h destroyed its ability to stimulate DNA synthesis in vitro, whereas wild-type extract was not inactivated until incubated at that temperature for 3 h.
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