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Role of nucleotide excision repair in processing of O4-alkylthymines in human cells
J C Klein1, M J Bleeker, H C Roelen
1Division of Molecular Carcinogenesis, The Netherlands Cancer Institute, Amsterdam.
Abstract:
O4-Alkylthymines have been implicated as potential carcinogenic DNA lesions. We have studied the effects of O4-methylthymine, O4-ethylthymine, and O4-n-propylthymine in a model system in which a single lesion was located at a defined position on a SV40-based shuttle vector and have found large differences in the effects of these lesions in repair-proficient and nucleotide excision repair-deficient cells. In repair-competent human HeLa cells, normal fibroblasts, and XP-A (2OS) revertant cells, all 3 residues were highly mutagenic; a mutation frequency of approximately 20% was found for both O4-methylthymine and O4-ethylthymine, whereas that of O4-n-propylthymine was approximately 12%. These frequencies were independent of the activity of the O6-alkylguanine DNA alkyltransferase. All three O4-alkylthymines induced T-->C transitions exclusively. In nucleotide excision repair-deficient XP-A cells, however, these lesions were not mutagenic but strongly inhibited plasmid replication (> 90%). These results indicate that O4-alkylthymines are efficiently recognized by the nucleotide excision repair system and cause a complete cessation of plasmid replication if this system is deficient. Nevertheless, proficiency in the nucleotide excision repair pathway correlates with a high frequency of mutation induction by these lesions.
Insights
O4-alkylthymines are mutagenic DNA lesions that cause T to C transitions in repair-proficient cells. However, these lesions inhibit DNA replication in nucleotide excision repair-deficient cells.
Area of Science:
- Molecular Biology
- DNA Repair Mechanisms
- Carcinogenesis
Background:
- O4-alkylthymines are DNA adducts recognized as potential carcinogenic lesions.
- Understanding the biological impact of these lesions is crucial for assessing their role in cancer development.
Purpose of the Study:
- To investigate the mutagenic and replication effects of O4-methylthymine, O4-ethylthymine, and O4-n-propylthymine.
- To compare the effects of these lesions in repair-proficient versus nucleotide excision repair-deficient cellular models.
Main Methods:
- A model system using a single O4-alkylthymine lesion at a defined position on an SV40-based shuttle vector.
- Analysis of mutation frequencies and plasmid replication in human HeLa cells, normal fibroblasts, and XP-A cells (nucleotide excision repair-deficient).
Main Results:
- In repair-proficient cells, all three O4-alkylthymines were highly mutagenic, inducing exclusively T to C transitions.
- Mutation frequencies were approximately 20% for O4-methylthymine and O4-ethylthymine, and 12% for O4-n-propylthymine, independent of O6-alkylguanine DNA alkyltransferase activity.
- In nucleotide excision repair-deficient XP-A cells, these lesions were non-mutagenic but strongly inhibited plasmid replication (>90%).
Conclusions:
- O4-alkylthymines are efficiently recognized and processed by the nucleotide excision repair system.
- Deficiency in nucleotide excision repair leads to a complete halt in plasmid replication upon encountering O4-alkylthymines.
- Nucleotide excision repair proficiency correlates with a high frequency of mutation induction by these DNA lesions.