The interplay between different repair mechanisms in human cells has long been a subject of significant interest. We provide a direct demonstration that in intact human cells the non-bulky oxidatively generated 5-guanidinohydantoin (Gh) and diastereomeric spiroiminodihydantoin (S,R-Sp) lesions, which are known to be repaired by base excision repair (BER), are also substrates of nucleotide excision repair (NER). The 32P-internally labeled double-stranded DNA substrates harboring the lesions were transfected into fibroblasts or HeLa cells. After 2-8 h incubation, the transfected cells were lysed and the 32P-labeled BER and NER early incision and dual excision repair products, respectively, were recovered and quantified by electrophoretic gel autoradiography methods. A bulky benzo[a]pyrene-derived guanine adduct (positive control for NER) yielded similar yields of NER products, but no BER products, as expected. After 4 h incubation periods, the NER yields in the case of the Sp and Gh lesions were ~ 4% and the ratios of [BER]/[NER] incision yields were ~ 5 (Gh) and ~10 (Sp). In human fibroblasts deficient in the key NER factor, XPA, the Sp and Gh lesions were repaired almost exclusively by BER pathways, and the NER ladders of dual excision products were not observed. This approach provides a convenient method for a real-time evaluation of the DNA repair capacities of human cells with different repair backgrounds by simultaneously monitoring the early BER and NER incision events.

Base and nucleotide excision repair of site-specific oxidatively generated guanine lesions in DNA substrates transfected into human cells

256th National Meeting (2018)

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