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Chemical and Biological Studies of DNA Carboxymethylation

  • Author(s): Wang, Jianshuang
  • Advisor(s): Wang, Yinsheng
  • et al.
Abstract

Chemical modification of DNA is of interest in toxicology and in evaluating human health risks associated with exposure to chemical carcinogens. Formation of various DNA adducts is considered an initial step for chemical species to exert their genotoxicity. DNA carboxymethylation has been suggested to be an important etiological factor in development of gastrointestinal cancers. In this dissertation, I focused on developing synthetic methods for preparation of novel carboxymethylated DNA lesions and incorporation of these lesions into oligodeoxyribonucleotides (ODNs) for biological studies. I also developed an LC-MS-based analytical method for identification of carboxymethylated DNA lesions formed in isolated DNA and in cultured human cells. The mutagenic and cytotoxic properties of these lesions in vivo were assessed by a newly developed next-generation sequencing (NGS) method.

Chapters 2 and 3 described the synthetic methods developed for synthesizing four novel carboxymethylated DNA lesions: N4-carboxymethyl-2'-deoxycytidine (N4-CMdC), N3-carboxymethylthymidine (N3-CMdT), O4-carboxymethylthymidine (O4-CMdT) and N6-carboxymethyl-2'-deoxyadenosine (N6-CMdA). By using LC-MS/MS, I confirmed that these lesions could be induced in calf thymus DNA treated with potassium diazoacetate (KDA). I also extended these synthetic methods to the preparation of lesion-carrying phosphoramidite derivatives, which allowed for the site-specific incorporation of these lesions into ODNs, and confirmed the identities of these ODNs by tandem mass spectrometry. Chapter 4 described the synthesis of stable isotope-labeled, carboxymethylated nucleoside derivatives which could serve as internal standards for future quantification study. With the use of these standards and LC-MS/MS, I found that O4-CMdT and N6-CMdA could be induced in human kidney epithelial cells (HEK 293T/17) upon exposure to azaserine. In Chapter 5, we developed a high-throughput method for examining the mutagenic and cytotoxic properties of carboxymethylated DNA lesions in cells. Our results illustrated that N3-CMdT induces predominantly T→A transversion mutation while O4-CMdT induces T→C transition mutation. Taken together, the combined chemical and biological approaches developed in this dissertation improved significantly our understanding of the biological implications of DNA carboxymethylation.

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