Numerous environmental and endogenous chemicals lead to the formation of addition products, or adducts, of DNA bases in the genome. While the cell has evolved DNA-repair pathways to remove such adducted DNA bases it is often the case that subsets of adducted bases are slowly repaired and can be detected in DNA years after the exposure that lead to their formation. Persistent DNA adducts may represent an ongoing danger to the tissues in which they are located, perhaps by causing mutations that contribute to tumor formation. Examples of exposures that lead to persistent DNA-adducts include components of tobacco smoke such as 3-nitrobenzanthrone, chemotherapeutic agents such as cisplatin, and a plant nephrotoxin and carcinogen found in many traditional Chinese medicines, aristolochic acid. The goal of this R21 proposal is to develop reagents and methods that allow for the isolation of genomic DNA fragments that contain DNA adducts and the deep sequencing of those DNAs to identify genomic regions containing such adducts. To achieve this goal, antibodies with high-specificity and affinity for the adducted base will be used to isolate DNA fragments with adducts from genomic DNA. To develop the method we will use antibodies specific for the aristolactam adduct formed after aristolochic acid exposures. A second goal is to identify the specific base that is adducted. The key to this method is to introduce a specific cleavage of the genomic DNA relative to the adducted base to allow the precise location of the adducted base in the sequenced DNA to be determined. To achieve this goal our antibodies will be fused to a DNA- endonuclease to create an adduct-specific nuclease. We will determine if the sequences that harbor repair- resistant adducts are the same that are mutated with high-frequency in tumors caused by aristolochic acid. The results of the proposed work should be the first genome-wide adductome compiled that links sequence information with adduct concentration. The specific sequences that escape the global genome repair mechanism will be of interest in the refinement of our understanding of life-long risks of environmental exposures.
Chemically modified DNA bases results from the exposure to many environmental agents and lead to pathological mutations that drive tumor formation. Even in cells with normal DNA-repair capacity some of these DNA-adducts persist and can remain for decades in the DNA of target organs. We propose to develop methods to determine the DNA sequences that harbor such persistent adducts.
