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Increased sensitivity to DNA-alkylating agents in CHO mutants with decreased poly(ADP-ribose) polymerase activity
M V Witmer1, N Aboul-Ela, M K Jacobson
1Lankenau Medical Research Center, Wynnewood, PA 19096.
Abstract:
Using a replica-plating procedure and a 32P-NAD+ permeable cell-screening assay, we have isolated a CHO mutant, PADR-9, which displays approximately 17% of the wild-type level of poly(ADP-ribose) polymerase activity. Biochemical analysis of the mutant using activity, Western, and Northern blot techniques indicate that relative to its parent cell, the mutant's enzyme activity, antibody recognition, and mRNA levels have been reduced to approximately the same extent. These results are consistent with a mutation in the PADR-9 cell which has resulted in a reduction in enzyme synthesis due to reduced mRNA synthesis and/or stability. Relative to wild-type CHO cells, the PADR-9 mutant has increased sensitivity to killing by DNA-alkylating agents but has normal gamma-ray sensitivity. Correlation between a decrease in poly(ADP-ribose) polymerase activity and an increased sensitivity to DNA-alkylating agents suggests that poly(ADP-ribose) synthesis may be important in the repair and/or induction of DNA damage produced by these agents.
Insights
Researchers isolated a CHO mutant, PADR-9, with reduced poly(ADP-ribose) polymerase activity. This mutant shows increased sensitivity to DNA-alkylating agents, suggesting the enzyme
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Poly(ADP-ribose) polymerase (PARP) is a crucial enzyme involved in DNA repair.
- Understanding PARP's role requires studying mutants with altered enzyme activity.
- CHO cells provide a well-characterized model system for genetic studies.
Purpose of the Study:
- To isolate and characterize a Chinese Hamster Ovary (CHO) cell mutant with deficient poly(ADP-ribose) polymerase (PARP) activity.
- To investigate the impact of reduced PARP activity on cellular sensitivity to DNA-damaging agents.
- To explore the relationship between PARP activity and DNA repair mechanisms.
Main Methods:
- Utilized replica-plating and a 32P-NAD+ permeable cell-screening assay to isolate the mutant.
- Performed biochemical analyses including enzyme activity assays, Western blots, and Northern blots.
- Assessed cellular sensitivity to DNA-alkylating agents and gamma-ray irradiation.
Main Results:
- Isolated CHO mutant PADR-9 exhibiting approximately 17% of wild-type poly(ADP-ribose) polymerase activity.
- Biochemical analyses revealed reduced enzyme activity, antibody recognition, and mRNA levels in PADR-9 cells.
- PADR-9 mutant displayed increased sensitivity to DNA-alkylating agents but normal sensitivity to gamma rays.
Conclusions:
- The mutation in PADR-9 likely reduces poly(ADP-ribose) polymerase synthesis via decreased mRNA synthesis and/or stability.
- Reduced poly(ADP-ribose) polymerase activity correlates with increased sensitivity to DNA-alkylating agents.
- Poly(ADP-ribose) synthesis appears critical for the repair or induction of DNA damage caused by alkylating agents.