Chemical Synthesis, Characterization and Biological Evaluation of Methylation and Glycation DNA Adducts

Qi Tang, University of Rhode Island


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 oligonucleotide syntheses: (1) purify oligonucleotides by different HPLC systems; (2) determine exact molecular weight by high-resolution MS; (3) locate modification position by MS/MS or exonuclease digestion with matrix-assisted laser desorption ionization-time of flight analysis; and (4) conduct, where applicable, enzymatic assays. We applied these steps to characterize byproducts in the syntheses of oligonucleotides containing biologically important methyl DNA adducts 1-methyladenine and 3-methylcytosine. In 1-methyladenine synthesis, we differentiated a regioisomeric byproduct 6-methyladenine, which possesses a molecular weight identical to uncharged 1-methyladenine. As for 3-methylcytosine, we identified a deamination byproduct 3-methyluracil, which is only 1 Da greater than uncharged 3-methylcytosine in the ?4900 Da context. The detection of these byproducts would be very challenging if the abovementioned procedure was not adopted. In MANUSCRIPT-II, as we have developed a platform enabling us to conduct in vitro synthesis and quality control of interested DNA adducts, we synthesized a number of N-methyl DNA adducts occurring at the Watson-Crick base-pairing face of the four nucleobases including 1-methyladenine, 3-methylcytosine, 1-methylguanine, and 3-methylthymine. We evaluated the repair preference of AlkB family DNA repair enzymes on these N-methyl DNA adducts under difference strand context. The AlkB protein is a repair enzyme that uses an ?-ketoglutarate/Fe(II)-dependent mechanism to repair alkyl DNA adducts. AlkB has been reported to repair highly susceptible substrates, such as 1-methyladenine and 3-methylcytosine, more efficiently in single strand-DNA than in double strand-DNA. Here, we tested the repair of weaker AlkB substrates 1-methylguanine and 3-methylthymine and found that AlkB prefers to repair them in double strand-DNA. We also discovered that AlkB and its human homologues, ALKBH2 and ALKBH3, are able to repair the aforementioned adducts when the adduct is present in a mismatched base pair. These observations demonstrate the strong adaptability of AlkB toward repairing various adducts in different environments. In MANUSCRIPT-III, we aimed to explore the potential interfering effects of the overconsumption of substance from environmental resources to DNA repair. We studied the inhibitory activity of a class of natural products, hydrolysable tannins, on ALKBH2 enzyme. [Shortened by ProQuest]

Subject Area

Biochemistry|Medicine|Pharmaceutical sciences

Recommended Citation

Qi Tang, "Chemical Synthesis, Characterization and Biological Evaluation of Methylation and Glycation DNA Adducts" (2019). Dissertations and Master's Theses (Campus Access). Paper AAI13858734.