TY - JOUR
T1 - Excision of Oxidatively Generated Guanine Lesions by Competing Base and Nucleotide Excision Repair Mechanisms in Human Cells
AU - Shafirovich, Vladimir
AU - Kropachev, Konstantin
AU - Kolbanovskiy, Marina
AU - Geacintov, Nicholas E.
N1 - Funding Information:
*E-mail: vs5@nyu.edu. Tel.: (212) 998-8456. ORCID Vladimir Shafirovich: 0000-0001-8225-6509 Funding This work was supported by the National Institute of Environmental Health Sciences grant R01 ES-027059 to V.S. Components of this work were conducted in the Shared Instrumentation Facility at New York University that was constructed with support from Research Facilities Improvement grant C06 RR-16572 from the National Center for Research Resources, National Institutes of Health (USA). Notes The authors declare no competing financial interest.
Publisher Copyright:
© 2019 American Chemical Society.
PY - 2019/4/15
Y1 - 2019/4/15
N2 - 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, 32 P-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.
AB - 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, 32 P-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.
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U2 - 10.1021/acs.chemrestox.8b00411
DO - 10.1021/acs.chemrestox.8b00411
M3 - Article
C2 - 30688445
AN - SCOPUS:85061489627
VL - 32
SP - 753
EP - 761
JO - Chemical Research in Toxicology
JF - Chemical Research in Toxicology
SN - 0893-228X
IS - 4
ER -