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DNA polymerase beta is required for efficient DNA strand break repair induced by methyl methanesulfonate but not by
P Fortini1, B Pascucci, F Belisario
1Laboratory of Comparative Toxicology and Ecotoxicology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.
Abstract:
The most frequent DNA lesions in mammalian genomes are removed by the base excision repair (BER) via multiple pathways that involve the replacement of one or more nucleotides at the lesion site. The biological consequences of a BER defect are at present largely unknown. We report here that mouse cells defective in the main BER DNA polymerase beta (Pol beta) display a decreased rate of DNA single-strand breaks (ssb) rejoining after methyl methanesulfonate damage when compared with wild-type cells. In contrast, Pol beta seems to be dispensable for hydrogen peroxide-induced DNA ssb repair, which is equally efficient in normal and defective cells. By using an in vitro repair assay on single abasic site-containing circular duplex molecules, we show that the long-patch BER is the predominant repair route in Pol beta-null cell extract. Our results strongly suggest that the Pol beta-mediated single nucleotide BER is the favorite pathway for repair of N-methylpurines while oxidation-induced ssb, likely arising from oxidized abasic sites, are the substrate for long-patch BER.
Insights
DNA polymerase beta (Pol beta) is crucial for repairing N-methylpurine DNA damage via single nucleotide base excision repair (BER). However, it is not essential for repairing oxidative DNA single-strand breaks, which are handled by long-patch BER.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Base excision repair (BER) is vital for maintaining mammalian genome integrity.
- Multiple BER pathways exist, but the consequences of defects, especially in DNA polymerase beta (Pol beta), are not fully understood.
Purpose of the Study:
- To investigate the role of DNA polymerase beta (Pol beta) in different base excision repair (BER) pathways.
- To elucidate the biological consequences of Pol beta deficiency in DNA repair.
Main Methods:
- Comparison of DNA single-strand break (ssb) rejoining rates in wild-type and Pol beta-defective mouse cells after methyl methanesulfonate (MMS) and hydrogen peroxide (H2O2) exposure.
- In vitro repair assays using abasic site-containing DNA substrates to analyze BER pathway preference.
Main Results:
- Pol beta-null cells showed reduced DNA ssb rejoining after MMS damage compared to wild-type cells.
- Pol beta-null cells exhibited normal DNA ssb repair efficiency after H2O2 exposure.
- In vitro assays revealed long-patch BER as the predominant pathway in Pol beta-null cell extracts.
Conclusions:
- Pol beta is the primary polymerase for single-nucleotide BER, essential for repairing N-methylpurine lesions.
- Oxidation-induced DNA ssb are repaired via the long-patch BER pathway, independent of Pol beta.