Date of Award

2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Pharmaceutical Sciences

Department

Biomedical and Pharmaceutical Sciences

First Advisor

Deyu Li

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 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 α-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. Hydrolysable tannins are a class of polyphenolic compounds commonly found in many plants. In this work, we studied the in vitro inhibitory mechanism of six molecules on ALKBH2. We determined the IC50 values of these compounds on the repair of 3-methylcytosine, the proto-typical substrate of ALKBH2. A structure-activity relationship was also observed between the strength of inhibition and the number of galloyl moieties in a molecule. In addition, we found that the inhibition by this class of polyphenolic compounds on ALKBH2 is through an iron-chelating mechanism. Direct reversal of alkyl DNA adducts by AlkB family enzymes constitute an important cellular repair mechanism for maintaining genome integrity. We here demonstrated that the potential effect of overdosing hydrolysable tannins might lead to the inhibition of iron-dependent AlkB family enzymes.

In MANUSCRIPT-IV, the objective of this work was to explore the in vitro synthesis and the in cell biological outcomes of glycation DNA adducts generated from cellular reducing sugar glucose-6-phosphate. We chemically synthesized and characterized the Amadori glycation products from glucose-6-phosphate and evaluated their toxic and mutagenic properties in cell. Reducing sugars and their metabolic derivatives (e.g. D-glucose, D- glucose-6-phosphate, methylglyoxal, and glyoxal) can non-enzymatically react with cellular biomacromolecules, such as proteins and nucleic acids, to induce glycation. Glucose and glucose-6-phosphate have been shown to glycate lysine residues of protein to form Amadori adducts. As a glycating agent with multiple-fold higher reactivity and much more abundant cellular concentration than glucose, glucose-6-phosphate has also been proposed to glycate DNA and cause genotoxicity with the formation of Amadori adducts. Due to the unstable nature of Amadori DNA adducts and a number of potential repair pathways, it is challenging to study their toxic and mutagenic properties in cell. Therefore, the specific biological outcomes of these adducts remain unclear. In this work, we used a number of chemical and genetic approaches and studied the biological consequences, such as replication block and mutations, of Amadori DNA adducts in a site-specific manner. Our data showed that Amadori DNA adducts arising from glucose-6-phosphate can induce G to T and single nucleotide deletion, which may lead to devastating biological consequences if left unrepaired, thereby affecting the genome integrity of organisms.

In summary, this dissertation offers a systematic strategy to conduct both in vitro and in cell studies of DNA adducts and mutagenesis. The methods applied in this work will not only broaden the basic research for chemical modification of nucleic acids, but also bring new aspects for studying DNA biology.

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