Abstract

The integrity of genomic DNA is constantly challenged by endogenous and environmental agents with the formation of DNA adducts. Some of these adducts are toxic and mutagenic to replication, thus potentially leading to cancer and other genetic diseases. To counteract the undesired DNA modifications from damaging agents, cells have evolved a number of repair pathways, such as base-excision repair, nucleotide-excision repair, mismatch repair, and direct reversal repair, to restore the intact DNA. However, DNA repair is not always efficient, there are many interfering factors, such as inherited deficiency in DNA repair pathways, or the abnormal uptake of substance with inhibitory effects to DNA repair enzymes that may result in the accumulation of DNA adducts. In the meantime, cells have equipped with mechanisms to carry out translesion bypass of DNA adducts, and the bypass of these adducts may lead to mutagenesis. Therefore, studying the DNA adduct formation, repair, and other mutagenic consequences can shed light on how DNA damaging agents impact on cellular response of organisms. A comprehensive understanding of the biological outcomes of interested DNA adducts requires the development of a set of efficient chemical approaches to prepare and characterize adduct-containing DNA oligonucleotides. On the other hand, the biological evaluation of these DNA adducts from various aspects, such as studying their genotoxic effects, and exploring potential interfering factors to their cellular repair, is essential for providing insights into the etiology of many diseases including cancer. This dissertation describes the chemical synthesis, characterization, and biological evaluation of methylation and glycation DNA adducts by using a variety of chemical and genetic tools. These strategies along with the findings obtained from the application of them are briefly discussed in the abstract of four manuscripts as following, and are described in detail in CHAPTER 1, 2, 3, and 4. In MANUSCRIPT-I, the objective of this study was to develop a rigorous procedure to chemically synthesize and characterize adduct-containing DNA oligonucleotides for biological studies. Oligonucleotides serve as important tools for biological, chemical, and clinical research. The preparation of oligonucleotides through automated solid-phase synthesis is well-established. However, identification of byproducts generated from DNA synthesis, especially from oligonucleotides containing site-specific modifications, is sometimes challenging. Typical high-performance liquid chromatography, mass spectrometry, and gel electrophoresis methods alone are not sufficient for characterizing unexpected byproducts, especially for those having identical or very similar molecular weight to the products. We developed a rigorous quality control procedure to characterize byproducts generated during

Highlights

  • Oligonucleotides synthesized chemically are widely used as drugs and research tools in biology, chemistry, and medicine

  • High resolution electrospray ionization (ESI)-TOF mass spectrometry (MS) analysis of the sample exhibited m/z at 1224.715 at its -4 charge state, which is in good agreement with the theoretical m/z 1224.711 expected of the product oligonucleotide (Figure 3a, S1 and Table 1)

  • The field of oligonucleotide synthesis has progressed such that DNA and RNA containing standard bases can be ordered from commercial sources

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Summary

Introduction

Oligonucleotides synthesized chemically are widely used as drugs and research tools in biology, chemistry, and medicine. If the lesions occur in a double stranded (ds) context, they may change local DNA architecture, leading to an unstable DNA duplex, or possibly disrupt the proper recognition of specific sites by sequence-specific DNA binding proteins, such as transcription factors.[1,2] To avoid these adverse effects from DNA adducts, organisms have developed an array of DNA repair pathways that are able to protect cells against lesions in both ss- and ds-DNA. We focus on the DNA repair enzyme ALKBH2 in the AlkB family, which use Fe(II)/αKG-dependent mechanism to repair alkyl nucleic acid lesions induced by endogenous and environmental alkylating agents (Figure 1a).[6,7]. Among the three amino group-containing nucleobases, guanine is most vulnerable to glycating agents; and N2-dG adducts are the primary source of DNA glycation products.[2,5]

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