LC-MS/MS for Assessing the Incorporation and Repair of N 2 -Alkyl-2'-deoxyguanosine in Genomic DNA.

Understanding the occurrence, repair, and biological consequences of DNA damage is important in environmental toxicology and risk assessment. The most common way to assess DNA damage elicited by exogenous sources in a laboratory setting is to expose cells or experimental animals with chemicals that modify DNA. Owing to the lack of reaction specificities of DNA damaging agents, the approach frequently does not allow for induction of a specific DNA lesion. Herein, we employed metabolic labeling to selectively incorporate N ² -methyl-dG ( N ² -MedG) and N ² - n -butyl-dG ( N ² - n BudG) into genomic DNA of cultured mammalian cells, and investigated how the levels of the two lesions in cellular DNA are modulated by different DNA repair factors. Our results revealed that nucleotide excision repair (NER) exert moderate effects on the removal of N ² -MedG and N ² - n BudG from genomic DNA. We also observed that DNA polymerases κ and η contribute to the incorporation of N ² -MedG into genomic DNA and modulate its repair in human cells. In addition, loss of ALKBH3 resulted in higher frequencies of N ² -MedG and N ² - n BuG incorporation into genomic DNA, suggesting a role of oxidative dealkylation in the reversal of these lesions. Together, our study provided new insights into the repair of minor-groove N ² -alkyl-dG lesions in mammalian cells.