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BER, MGMT, and MMR in defense against alkylation-induced genotoxicity and apoptosis
1Division of Applied Toxicology, Institute of Toxicology, University of Mainz, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany.
Abstract:
Methylating carcinogens and cytostatic drugs induce different methylation products in DNA. In cells not expressing the repair protein MGMT or expressing it at a low level, O6-methylguanine is the major genotoxic, recombinogenic, and apoptotic lesion. Genotoxicity and apoptosis triggered by O6-methylguanine require mismatch repair (MMR). In cells expressing O6-methylguanine-DNA methyl transferase (MGMT) at a high level or for agents producing low amounts of O6-methylguanine, N-alkylations become the major genotoxic lesions. N-Alkylations are repaired by base excision repair (BER). In mammalian cells, naturally occurring mutants of BER have not been detected, which points to the importance of BER for viability. In order to ascertain the role of BER in cellular defense, BER was modulated either by transfection or mutational inactivation. It has been shown that overexpression of N-methylpurine-DNA glycosylase (MPG) does not protect, but rather sensitizes cells to SN2 agents. This has been interpreted in terms of an imbalance in BER. Regarding abasic site endonuclease (APE), transient but not stable overexpression of the enzyme was achieved upon transfection in CHO cells, which indicates that unphysiologic APE levels are not tolerated by the cell. Besides the repair function, APE (alias Ref-1) exerts redox capability by which the activity of various transcription factors is modulated. Therefore, it is possible that stable overexpression of mammalian APE impairs transcriptional regulation of genes, whereas transient overexpression may exert some protective effect. DNA polymerase beta (Pol beta) transfection was ineffective in conferring resistance to methylmethane sulfonate (MMS). On the other hand, Pol beta-deficient cells proved to be highly sensitive to methylation-induced chromosomal aberrations and reproductive cell death. The dramatic hypersensitivity in the killing response is largely due to induction of apoptosis. Obviously, nonrepaired BER intermediates are clastogenic and act as a strong trigger of the apoptotic pathway. The elements of this pathway are currently under investigation.
Insights
DNA repair pathways are crucial for cellular defense against methylating agents. Base excision repair (BER) intermediates trigger apoptosis when not properly repaired, highlighting BER
Area of Science:
- Molecular Biology
- DNA Repair Mechanisms
- Carcinogenesis
Background:
- Methylating agents induce DNA damage, forming lesions like O6-methylguanine and N-alkylations.
- DNA repair pathways, including MGMT, MMR, and BER, counteract these lesions.
- The roles of specific BER enzymes in cellular defense against genotoxic agents are not fully understood.
Purpose of the Study:
- To investigate the role of Base Excision Repair (BER) in cellular defense against methylating agents.
- To determine the impact of modulating BER enzyme levels on cellular sensitivity to DNA damage.
- To elucidate the mechanisms by which BER intermediates trigger apoptosis.
Main Methods:
- Modulation of BER enzyme expression (MPG, APE, Pol beta) through transfection and mutational inactivation in mammalian cells (CHO cells).
- Assessment of cellular sensitivity to methylating agents (SN2 agents, MMS).
- Analysis of genotoxicity, apoptosis induction, and chromosomal aberrations.
- Investigation of abasic site endonuclease (APE) function, including its repair and redox capabilities.
Main Results:
- Overexpression of N-methylpurine-DNA glycosylase (MPG) sensitized cells to SN2 agents, suggesting BER imbalance.
- Stable overexpression of abasic site endonuclease (APE) was not tolerated, while transient overexpression showed potential protective effects.
- DNA polymerase beta (Pol beta)-deficient cells exhibited extreme sensitivity to methylation-induced damage and apoptosis.
- Unrepaired BER intermediates were identified as clastogenic and potent inducers of apoptosis.
Conclusions:
- BER plays a critical role in protecting cells from methylation-induced DNA damage.
- Aberrations in BER enzyme levels can lead to cellular sensitization and increased genotoxicity.
- BER intermediates are key triggers of the apoptotic pathway in response to DNA damage.
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