DNA damage drives human genetic disease including cancer. Exogenous chemicals and endogenous reactive molecules can damage DNA bases forming ?adducts?. Unrepaired DNA adducts can block DNA synthesis or miscode during polymerase-mediated replication. The landscape of mutations observed in human cancers is dominated by C:G to T:A transition mutations. A major cause of these mutations is the hydrolytic deamination of cytosine and cytosine analogs in DNA to their corresponding uracil analogs, generating a class of cytosine deamination adducts, xU. Endogenous DNA adducts such as xU have proven more difficult to study due to the similarity of these adducts to normal DNA constituents as well as their formation in normal, unperturbed cells and tissues. In this application, we describe innovative new approaches that will allow definitive identification of xU adducts in DNA using mass spectrometry methods. Further, we will measure the formation and repair of such adducts at known cancer-driving mutational hotspots and genome-wide in both normal human cells and cells with known repair defects. We will also examine xU in discard human tissues representing normal, inflamed and diseased specimens. The studies proposed here will provide an unprecedented examination of this important but understudied class of DNA adducts at the level of DNA sequence. The results of the proposed studies could potentially result in clinically useful approaches to examine the damage history of a given tissue, and provide an estimate as to how far the damage had progressed toward the development of tumors. The anticipated results will shed new light on cancer etiology and potentially direct approaches to reduce cancer incidence and provide earlier detection. ! !
In this application, we describe innovative new approaches using state-of-the-art methods to measure the types of DNA damage that cause the most common mutations in human cancers. We will measure the levels of DNA damage in normal cells, cancer cell lines and DNA from human tissues, as well as how genetic differences in DNA repair capacity influence the incidence of cancer-driving mutations. Ultimately, the proposed studies will significantly increase our understanding of how DNA damage results in cancer and could lead to new approaches to reduce cancer incidence and improve early diagnosis by providing new means to measure the ?damage history? of a tissue and to estimate how far the tissue has progressed toward tumor development. ! !
