Mass Spectrometric Studies of Structurally Modified DNA

The advances in mass spectrometry (MS) instrumentation have rendered MS an indispensable tool for structure elucidation, quantification, and assessment of the biological consequences of DNA modifications. DNA encodes genetic information that is used in the development and functioning of essentially all known organisms. Changes in DNA could give rise to compromised transmission of genetic information. Abnormal modifications are generally harmful to cells and will be converted to permanent genetic damage if they evade DNA repair, whereas epigenetic DNA modifications are regulators of cell function that influence chromatin structure and control the levels of gene expression. The emphasis of this dissertation is on the application of MS, together with biological techniques, for assessing the occurrence and biological consequences of these two major types of DNA modifications.After an introduction presented in Chapter 1, Chapter 2 focuses on monitoring the enzymatic removal of 8-methoxypsoralen-induced DNA interstrand cross-links (ICLs) in cultured mammalian cells by using an HPLC coupled with tandem-MS approach. With genetic depletion of DNA repair proteins, we also pinpointed the role of nucleotide excision repair pathway in removing DNA ICLs. Chapters 3 and 4 discuss the development of an HPLC-MS/MS/MS method, along with the isotope dilution technique, for accurate measurements of 5-hydroxymethyl, 5-formyl and 5-carboxyl derivatives of 2'-deoxycytidine and 5-hydroxymethyl-2'-deoxyuridine (5-hmdU) in genomic DNA of cultured human cells, mammalian tissues and plant tissues. This is the first direct quantitative comparison between the levels of multiple 5-mdC derivatives in mammalian tissues and the first report of their presence in the model plant Arabidopsis. Chapter 5 describes the combination of this method with other bioanalytical techniques for studying the mechanisms of arsenic epigenotoxicity. We found that arsenite could bind directly to the zinc fingers of Tet proteins and substantially impair the catalytic efficiency of Tet proteins in oxidizing 5-mC both in vitro and in cells. In Chapter 6, we report the application of LC-MS/MS for quantifying β-D-glucosyl-5-hydroxymethyl- 2'-deoxyuridine (dJ) in Trypanosoma brucei. The results provide evidence that dJ binding proteins play an important role in oxidizing thymidine to form 5-hmdU, which then facilitates the generation of dJ