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Two pathways for base excision repair in mammalian cells
G Frosina1, P Fortini, O Rossi
1Laboratory of Centro Studi Tumori Ambientali-Mutagenesis, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy.
The Journal of Biological Chemistry
|April 19, 1996
Summary
Mammalian cells repair DNA abasic sites via two pathways: direct single-nucleotide insertion or a proliferating cell nuclear antigen-dependent process involving a larger patch. This clarifies DNA lesion repair mechanisms.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Abasic sites (apurinic/apyrimidinic sites) are frequent DNA lesions from spontaneous and induced base loss.
- Previous studies showed efficient repair of multiple AP sites in hamster cell extracts with an average patch size of 6.6 nucleotides.
Purpose of the Study:
- To precisely define the repair patch size and mechanism for a single abasic site in mammalian DNA.
- To investigate the involvement of specific proteins, like PCNA, in abasic site repair.
Main Methods:
- Construction of a circular DNA duplex containing a single abasic site.
- In vitro repair assays using Chinese hamster and human cell extracts.
- Restriction endonuclease analysis to characterize repair synthesis.
- Inhibition studies using antibodies against proliferating cell nuclear antigen (PCNA).
Main Results:
- Two distinct repair pathways were identified for abasic sites.
- Pathway 1: Single nucleotide incorporation directly at the lesion site.
- Pathway 2: A PCNA-dependent pathway involving a ~7 nucleotide repair patch 3' to the abasic site.
Conclusions:
- Mammalian cells employ dual mechanisms for abasic site repair.
- The PCNA-dependent pathway represents a novel mechanism for repairing DNA lesions and flanking nucleotides.