The carboxyl-terminal one-third of human topoisomerase II polypeptide expressed in Escherichia coli was used as antigen to generate polyclonal antibodies in rabbits. With the use of antiserum, DNA topoisomerase II levels of phytohemagglutinin-stimulated human lymphocytes were measured by immunoblotting. Our results showed that the increase in intracellular topoisomerase II level paralleled the entry of cells into proliferation. We also found that the increase in the topoisomerase II level resulted from an increase in the amount of topoisomerase II mRNA. The time course study indicated that the appearance of topoisomerase II mRNA was first observed at 36 h after phytohemagglutinin stimulation. The maximal level of topoisomerase II mRNA was seen at 45 h after stimulation. The same RNA blot was rehybridized with a thymidine kinase probe. The maximal level of thymidine kinase mRNA was observed at 39 h after phytohemagglutinin stimulation. In a comparison of the time course of topoisomerase II gene expression with that of [3H]-thymidine incorporation and thymidine kinase gene expression, it was found that the expression of the topoisomerase II gene was later than the onset of DNA replication. Thus, this study suggests that topoisomerase I, which is constantly expressed throughout the cell cycle, might participate in the initiation of DNA replication, while topoisomerase II is involved in solving the DNA topological problems accompanying DNA strand separation during DNA replication.
|出版狀態||已發佈 - 九月 1 1990|
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