Excision of Oxidatively Generated Guanine Lesions by Competing Base and Nucleotide Excision Repair Mechanisms in Human Cells

• Published in: Chemical research in toxicology Vol. 32; no. 4; pp. 753 - 761
• Main Authors: Shafirovich, Vladimir, Kropachev, Konstantin, Kolbanovskiy, Marina, Geacintov, Nicholas E
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 15.04.2019
• Subjects: Cells, Cultured; DNA Repair; Fibroblasts - metabolism; Guanine - chemistry; Guanine - metabolism; HeLa Cells; Humans; Kinetics; Molecular Structure; Oxidation-Reduction
• ISSN: 0893-228X; 1520-5010; 1520-5010
• DOI: 10.1021/acs.chemrestox.8b00411

The interchange between different repair mechanisms in human cells has long been a subject of interest. Here, we provide a direct demonstration that the oxidatively generated guanine lesions spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) embedded in double-stranded DNA are substrates of both base excision repair (BER) and nucleotide excision repair (NER) mechanisms in intact human cells. Site-specifically modified, 32P-internally labeled double-stranded DNA substrates were transfected into fibroblasts or HeLa cells, and the BER and/or NER mono- and dual incision products were quantitatively recovered after 2–8 h incubation periods and lysis of the cells. DNA duplexes bearing single benzo­[a]­pyrene-derived guanine adduct were employed as positive controls of NER. The NER activities, but not the BER activities, were abolished in XPA–/– cells, while the BER yields were strongly reduced in NEIL1–/– cells. Co-transfecting different concentrations of analogous DNA sequences bearing the BER substrates 5-hydroxyuracil diminish the BER yields of Sp lesions and enhance the yields of NER products. These results are consistent with a model based on the local availability of BER and NER factors in human cells and their competitive binding to the same Sp or Gh BER/NER substrates.