Abstract
DNA alkylation damage induced by environmental carcinogens, chemotherapy drugs, or endogenous metabolites plays a central role in mutagenesis, carcinogenesis, and cancer therapy. Base excision repair (BER) is a conserved, front line DNA repair pathway that removes alkylation damage from DNA. The capacity of BER to repair DNA alkylation varies markedly between different cell types and tissues, which correlates with cancer risk and cellular responses to alkylation chemotherapy. The ability to measure cellular rates of alkylation damage repair by the BER pathway is critically important for better understanding of the fundamental processes involved in carcinogenesis, and also to advance development of new therapeutic strategies. Methods for assessing the rates of alkylation damage and repair, especially in human cells, are limited, prone to significant variability due to the unstable nature of some of the alkyl adducts, and often rely on indirect measurements of BER activity. Here, we report a highly reproducible and quantitative, cell-based assay, named alk-BER (alkylation Base Excision Repair) for measuring rates of BER following alkylation DNA damage. The alk-BER assay involves specific detection of methyl DNA adducts (7-methyl guanine and 3-methyl adenine) directly in genomic DNA. The assay has been developed and adapted to measure the activity of BER in fungal model systems and human cell lines. Considering the specificity and conserved nature of BER enzymes, the assay can be adapted to virtually any type of cultured cells. Alk-BER offers a cost efficient and reliable method that can effectively complement existing approaches to advance integrative research on mechanisms of alkylation DNA damage and repair.
| Original language | English (US) |
|---|---|
| Article number | 18393 |
| Journal | Scientific reports |
| Volume | 11 |
| Issue number | 1 |
| DOIs | |
| State | Published - Dec 2021 |
Bibliographical note
Funding Information:We thank Dr. Ann Bode for help with proof reading the manuscript. We would like to acknowledge Dr. John Wyrick for helpful discussions and intellectual contributions to this work. This work was supported by grant R01ES002614 from the National Institute of Environmental Health Sciences (NIEHS) (to M.J.S. and J.J.W.), R21ES028549 from NIEHS to W.C., and Faculty Research Grant from University of Georgia to W.C. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS, NIH.
Publisher Copyright:
© 2021, The Author(s).
