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Poly(ADP ribose) polymerase-defective mutant cell clone of mouse L1210 cells
K Yoshihara1, A Itaya, T Hironaka
1Department of Biochemistry, Nara Medical University, Japan.
Abstract:
By a sequential mutation and selection utilizing N-methyl-N'-nitro-N-nitrosoguanidine as a mutagen, we succeeded in separating a poly(ADP ribose) polymerase-defective mutant clone (Cl-3527) from a mouse L1210 cell clone (Cl-3). The enzyme activity per cell in Cl-3527 cells was only 8% of that in wild type L1210 (CCL 219) cells. Immunoblot analysis of the enzyme protein in crude extracts of the mutant and wild type cells revealed that the enzyme defect was manifested as the loss of a 113-kDa wild type enzyme band in Cl-3527. Further analysis of partially purified enzyme from Cl-3527 by immunoblotting revealed that the molecular size of the enzyme in Cl-3527 was 108 kDa and that the amount of the mutant enzyme protein was markedly decreased in Cl-3527. The mutant enzyme was much more heat-labile than the wild type enzyme but the Km for NAD+, requirements for Mg2+ and nicked DNA, and the inhibition by 3-aminobenzamide, a potent inhibitor of the enzyme, however, were not so different from those of wild type enzyme. The mutant cells showed prolonged doubling time, increased temperature-sensitivity, increased percentage of active enzyme on a treatment of cells at high temperature, and increased expression of plasma membrane NADase, compared to wild type cells. Introduction of wild type ADPR pol gene into Cl-3527 cells partially restored the ADPR pol activity and the heat-resistance.
Insights
Researchers developed a poly(ADP-ribose) polymerase-defective mutant mouse L1210 cell clone (Cl-3527). This mutant exhibits significantly reduced enzyme activity and altered enzyme properties, impacting cell growth and heat resistance.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Poly(ADP-ribose) polymerase (ADPR pol) is crucial for DNA repair and cell survival.
- Understanding ADPR pol function requires the study of its mutants.
- Mouse L1210 cells are a well-established model for leukemia research.
Purpose of the Study:
- To generate and characterize a poly(ADP-ribose) polymerase-defective mutant of mouse L1210 cells.
- To investigate the biochemical and cellular consequences of ADPR pol deficiency.
- To explore the role of ADPR pol in cellular response to stress.
Main Methods:
- Sequential mutation and selection using N-methyl-N'-nitro-N-nitrosoguanidine.
- Enzyme activity assays.
- Immunoblot analysis and partial enzyme purification.
- Cellular phenotype analysis (doubling time, temperature sensitivity).
Main Results:
- A poly(ADP-ribose) polymerase-defective mutant clone (Cl-3527) was isolated with only 8% of wild-type enzyme activity.
- The mutant enzyme showed a reduced molecular size (108 kDa vs 113 kDa) and decreased protein levels.
- Mutant cells displayed prolonged doubling time, increased heat sensitivity, and altered enzyme characteristics.
- Introduction of the wild-type ADPR pol gene partially restored enzyme activity and heat resistance.
Conclusions:
- The generated mutant provides a valuable tool for studying poly(ADP-ribose) polymerase function.
- ADPR pol deficiency significantly impacts cell proliferation, DNA repair, and stress response.
- The study highlights the critical role of ADPR pol in maintaining cellular integrity and function.