Further characterization of the phenotype of ts20, a DNA ts mutant of BALB/3T3 cells

Abstract

Ts20 is a temperature-sensitive mutant cell line derived from BALB/3T3 cells that is blocked at a step in DNA synthesis involving chain elongation. Following a shift from 33 ° to 39 °C, mutant cells lost ability to grow or form colonies. When mutant cells were infected with polyomavirus, both cell and virus DNA synthesis were inhibited at the restrictive temperature of 39 °C. When cell extracts from wild-type cells were added in vitro to lysed infected mutant cells that had been incubated in vivo at 39 °C for expression of the mutation, cell DNA synthesis was increased 3-fold (similar to the effect in uninfected mutant cells), whereas virus DNA synthesis was increased only 60%. With harsher lysis conditions, the effect of added extract on virus DNA synthesis was greater, although baseline DNA synthesis (prior to addition of extracts) was much lower. Analysis by alkaline sucrose gradients showed that the addition of cell extract converted small cellular DNA molecules into larger ones, while it increased the synthesis of small virus DNA molecules rather than completed genomes. Analysis of cytosol extracts (in which the activity stimulating DNA synthesis resides) showed that DNA topo-isomerase I activity was more heat-labile when assayed in mutant extracts compared to wild-type extracts. In contrast, cytosol DNA polymerase activity was equally heat-labile in mutant and wild-type extract. This suggested the factor in extract was likely associated with the activity of DNA topo-isomerase I. Analysis of virus DNA synthesized in vitro in restricted mutant cells by gel electrophoresis and fluorography showed an accumulation of topo-isomers migrating between form I and II. These topo-isomers, thought to be a manifestation of the ts defect, did not disappear when extract from wild-type cells was added back in vitro or when mutant cells were shifted back to permissive temperature prior to lysis for in vitro synthesis. The results indicate that polyoma DNA synthesis and cell DNA synthesis differ in their response to the mutant gene product in ts20, although both are inhibited at a step early in DNA chain elongation that may involve DNA topo-isomerase I.