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Mutator phenotype in a mutant of S49 mouse T-lymphoma cells with abnormal sensitivity to thymidine
Abstract:
We have selected and characterized a thymidine-sensitive S49 mutant line, MC-3-3. MC-3-3 cells are 35-fold more sensitive to the cytotoxic effects of thymidine and 15-fold more sensitive to the cytotoxic effects of 5-bromodeoxyuridine than wild type S49 cells. In contrast, the MC-3-3 mutant line does not exhibit increased sensitivity to the cytotoxic action of 5-fluorodeoxyuridine. The MC-3-3 mutant line possesses levels of thymidylate synthetase and thymidine kinase activity which are equivalent to the levels in wild type S49 cells, but the ribonucleotide reductase activity in MC-3-3 cells, using CDP as a substrate, is only 10-30% of that in wild type cells. Using ADP as a substrate, the ribonucleotide reductase activity in permeabilized MC-3-3 cells is slightly higher than that in wild type S49 cells. The deoxyribonucleotide pools in exponentially growing MC-3-3 cells are approximately 40-50% of those in wild type S49 cells. By hybrid analysis, we determined that the thymidine sensitivity of the MC-3-3 cells is recessive. The MC-3-3 mutant line displays a rate of spontaneous mutation which is 15-30-fold higher than that of wild type S49 cells. The MC-3-3 mutant cells are also 5-10-fold more sensitive than wild type cells to the cytotoxic effects of tunicamycin and compactin. These results suggest that the MC-3-3 mutant line possesses a mutation in the dTTP binding site in ribonucleotide reductase; abnormal regulation of this enzyme results in an increase in the rate of spontaneous mutation.
Insights
A new S49 mutant cell line, MC-3-3, shows increased sensitivity to thymidine and 5-bromodeoxyuridine due to reduced ribonucleotide reductase activity. This defect elevates the spontaneous mutation rate, suggesting a mutation in the enzyme
Area of Science:
- Cell Biology
- Molecular Genetics
- Biochemistry
Background:
- S49 cell lines are widely used models for studying purine and pyrimidine metabolism.
- Thymidine sensitivity can indicate defects in DNA synthesis and repair pathways.
- Ribonucleotide reductase is a critical enzyme for deoxynucleotide synthesis, regulating cellular dNTP pools.
Purpose of the Study:
- To characterize a novel thymidine-sensitive S49 mutant cell line, MC-3-3.
- To investigate the underlying biochemical defect responsible for thymidine sensitivity.
- To determine the impact of this mutation on enzyme activity, deoxynucleotide pools, and spontaneous mutation rates.
Main Methods:
- Cytotoxicity assays with thymidine, 5-bromodeoxyuridine, and 5-fluorodeoxyuridine.
- Enzyme activity assays for thymidylate synthetase, thymidine kinase, and ribonucleotide reductase.
- Measurement of intracellular deoxyribonucleotide pools.
- Hybrid analysis to determine dominance/recessiveness of the mutation.
- Mutation rate determination and sensitivity assays for tunicamycin and compactin.
Main Results:
- MC-3-3 cells exhibit 35-fold higher sensitivity to thymidine and 15-fold higher sensitivity to 5-bromodeoxyuridine compared to wild-type S49 cells.
- Ribonucleotide reductase activity (using CDP as substrate) in MC-3-3 cells is significantly reduced (10-30% of wild-type), while thymidylate synthetase and thymidine kinase activities are normal.
- Deoxyribonucleotide pools are reduced by 40-50% in MC-3-3 cells.
- The thymidine sensitivity is recessive, and MC-3-3 cells show a 15-30-fold higher spontaneous mutation rate and increased sensitivity to tunicamycin and compactin.
Conclusions:
- The MC-3-3 mutant line likely harbors a mutation in the dTTP binding site of ribonucleotide reductase.
- Dysregulation of ribonucleotide reductase activity leads to altered deoxynucleotide pools and increased genomic instability.
- This mutant provides a valuable tool for studying DNA synthesis regulation and the link between enzyme defects and mutation rates.