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DNA-ligase activities appear normal in the CHO mutant EM9
Mutation Research
|May 1, 1984
Summary
Chinese hamster ovary (CHO) mutant EM9 cells, defective in DNA repair, were tested for DNA ligase defects. Results show EM9 cells do not have a primary defect in DNA ligase activity, despite altered levels of certain ligase forms.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The Chinese hamster ovary (CHO) mutant strain EM9 exhibits hypersensitivity to DNA damaging agents like ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS).
- EM9 cells show increased sister-chromatid exchanges (SCE) and are deficient in rejoining DNA strand breaks induced by various agents.
- A potential biochemical defect in DNA ligase activity was investigated as the cause of these phenotypes.
Purpose of the Study:
- To investigate whether a defect in DNA ligase activity is the primary biochemical cause of the observed DNA repair deficiencies in the EM9 CHO cell line.
- To compare DNA ligase activities in EM9 cells with those in the parental AA8 cell line and a phenotypic revertant (9R1).
Main Methods:
- DNA ligase activities were assayed in cellular extracts separated by AcA 34 gel-filtration chromatography.
- Levels of DNA ligases Ia, Ib, and II were quantified in EM9, AA8, and 9R1 cells.
- Cells were treated with methyl methanesulfonate (MMS) to assess the induction of DNA ligase activity following DNA damage.
- Ligase activity was further tested under conditions of high salt (0.1 M NaCl) and heat (50°C).
Main Results:
- EM9 cells displayed approximately 50% and 60% of parental AA8 levels for DNA ligases Ia and Ib, respectively; DNA ligase II levels were unaltered.
- The phenotypic revertant 9R1 also showed reduced levels of ligases Ia and Ib, suggesting these reductions are not directly linked to the mutant phenotype.
- MMS treatment caused a decrease in ligases Ia and Ib activity but a 2.0- and 2.6-fold increase in ligase II activity in AA8 and EM9 cells, respectively.
- No significant differences in ligase activity between EM9 and AA8 cells were observed under high salt or heat stress conditions.
Conclusions:
- The data strongly indicate that the primary defect in the EM9 mutant strain does not reside in DNA ligase activity.
- While altered levels of DNA ligases Ia and Ib are present in EM9 cells, they do not appear to be the cause of the DNA repair defect.