At the restrictive temperature of 39(DEGREES)C, DNA synthesis in ts20, a temperature-sensitive mutant of Balb/3T3 cells, decreases progressively after an initial increase during the first 3 h while RNA and protein synthesis remains at a high level for 40 h. Infection with polyomavirus does not bypass the defect in cell DNA synthesis and the mutant does not support virus DNA replication at 39(DEGREES)C. The synthesis of form I and replicative intermediates of virus DNA at 39(DEGREES)C decrease at about the same rate. The block to cell DNA synthesis is reversible while the block to virus DNA synthesis is not. The defects in virus and cell DNA synthesis are expressed in vitro as reduced ('3)H-dTTP incorporation, consistent with the in vivo data. The addition of wild-type cell extracts complements the depressed cell DNA synthesis in vitro. The complementary factor is detectable in cytosol and can be partially purified. Analysis of virus DNA synthesized in vitro in the mutant cells with ts phenotype reveals an accumulation of DNA molecules migrating between form I and form II. This indicates a topological block in the mutant. Both the complementing activity and DNA topoisomerase I activity are extractable into cytosols. In extracts from mutant cells both activities are present in lower amounts and both are thermolabile. Ts20 is a DNA('ts) mutant whose defect blocks an early step in DNA chain elongation and includes the loss of DNA topoisomerase I activity. These studies provide evidence for a role for this enzyme at this step in eukaryotic DNA replication.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.71983 |
| Date | January 1984 |
| Creators | Zeng, Guichao. |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Medicine.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 000214334, proquestno: AAINL20844, Theses scanned by UMI/ProQuest. |
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