DNA Adduct Detection and Repair in Mammalian Cells

PROJECT SUMMARY/ABSTRACT Numerous endogenous agents, environmental carcinogens, and anti-cancer drugs produce bulky base adducts in the genome. It is expected that the location of these adducts is non-random and that their locations dictate their pathogenic or therapeutic effects as well as their susceptibility to DNA repair enzymes that modulate these effects. The long-term goal of our research program is to better understand DNA damage and repair at the genomic level to aid in predicting and potentially preventing DNA adduct-induced carcinogenesis as well as to design efficient chemotherapeutic regimens with minimal side effects. The objective of this particular proposal is to use our novel adductomic-mapping methods (Damage-seq and XR-seq) to locate the exact positions of DNA lesions in the mammalian genome and the exact positions of DNA incisions performed by the nucleotide excision repair enzyme system that removes the DNA damage. The rationale for the proposed research is that mapping damage and repair may reveal unexpected links between environmental carcinogens, mutagenesis, disease and ageing. We will accomplish this goal by carrying out the following three specific aims: 1) Genomic Single- nucleotide Resolution Analysis of DNA Damage by Endogenous Agents; 2) Genome-wide Single Nucleotide Resolution Maps of DNA Damage and Repair by Exogenous Carcinogens; 3) Genome-wide Damage and Repair Maps of Anticancer Drugs. For this proposal, we will focus on identifying the precise locations and removal of DNA base damage formed endogenously (glycosylation, oxidative damage) and damage caused by environmental carcinogens (aflatoxin, diethylnitrosamine) and anticancer drugs (cisplatin, melphalan). Methods that we developed and further optimized for mapping damage formation and repair will be used throughout this work. This proposal is innovative because of these unique sequencing technologies that provide high-resolution DNA sequence information on the formation and repair of damage throughout the entire genome and provide an unparalleled approach for characterizing endogenous DNA damage as well as damage induced by environmental carcinogens and anti-cancer drugs. The proposed research is significant because it will address the question of the role of DNA damage by endogenous agents (glycosylation) of various tissues and its overall contribution to aging by these methods developed in our laboratory which have unprecedented sensitivity for genome-wide mapping at single nucleotide resolution in different organs. In addition, the proposed research will expand our understanding of DNA damage formation and repair in the human genome at an unprecedented level of detail regarding genomic damage formed endogenously and by environmental carcinogens and anticancer drugs. Ultimately, this knowledge has the potential to improve the prevention strategies for environmental carcinogenesis and to lead to the development of new tools for diagnosing and treating cancer.