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.
Highlights
DNA alkylation damage induced by environmental carcinogens, chemotherapy drugs, or endogenous metabolites plays a central role in mutagenesis, carcinogenesis, and cancer therapy
Activated poly ADP-ribose polymerase 1 (PARP1) catalyzes the formation of ADP-ribose chains, which serve as a docking platform that facilitates recruitment and assembly of the multiprotein Base excision repair (BER) complex (XRCC1-POLβ-LIG3)
We initially developed the alk-BER assay to measure BER in yeast cells (S. cerevisiae) and successfully adapted the assay to other fungal model organism (N. crassa) and human cells
Summary
DNA alkylation damage induced by environmental carcinogens, chemotherapy drugs, or endogenous metabolites plays a central role in mutagenesis, carcinogenesis, and cancer therapy. 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 to advance development of new therapeutic strategies. Persistent and inefficiently repaired methyl DNA adducts can induce microsatellite instability, frameshift mutations, and G → A transition mutations, that are commonly found in genes critical for malignant transformation, including the H-ras oncogene or TP53 tumor suppressor gene[3,4,5] Despite their carcinogenic properties, DNA alkylating agents, such as dacarbazine, temozolomide and streptozotin, have been used for decades in treating various cancers, including melanoma, glioma, and lymphoma[2,6,7]. Base excision repair (BER) is the primary pathway involved in the removal of alkylation DNA damage induced by methylating a gents[2,12,13]. Identifying a pre-existing BER imbalance within a tumor may be highly relevant for determining whether therapy involving PARP inhibitors and alkylating agents can be beneficial
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