Date of Award

1-1-2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Pharmaceutical Sciences

Department

Biomedical and Pharmaceutical Sciences

First Advisor

Deyu Li

Abstract

DNA is necessarily to be stable and intact since it is the repository of genetic information in each living cell. However, DNA is not inert, and it is vulnerable to the endogenous and environmental assault including reactive oxygen species (ROS), ultraviolet (UV) light, and various carcinogens. Exposure to these DNA damaging agents is associated with various adducts ranging from small methyl or etheno adducts to bulky adducts such as acetylaminofluorene. These resulting damages, if not repaired, will lead to replication block or mutation which can eventually cause cancer or other genetic diseases. The research covered by this dissertation falls into two main categories. One section focuses on in-cell investigations on the sequence-specific bypass and mutagenesis of bulky DNA lesions generated from 2-acetylaminofluorene (AAF), and the other section discusses how 2OG/Fe(II)-dependent dioxygenases, such as AlkB and TET family enzymes, repair etheno DNA adducts.

By developing a mass-spectrometry based system, we are able to test the bypass and mutagenesis of single AAF adduct in different flanking sequence combinations. The result showed that the epigenetic marker 5mC may play a different role than a canonical C in the flanking bulky lesion bypass, and It's also interesting to note that, regardless of whether the 5'C of the lesion site was methylated or not, only lesion bypass in the sequences CGA, CG1G, and CGG2 had the potential to produce a -G frameshift mutation.By studying the role of TET1 and ALKBH2 enzymes in repairing 3,N4-etheno-5-methylcytosine (ε5mC), the etheno adduct of the major epigenetic marker 5-methylcytosine (5mC), we found ε5mC cannot be oxidized by TET to its oxidative derivatives, such as 5hmC, 5fC and 5caC. However, ALKBH2 is able to oxidatively repair ε5mC to 5mC, which is subsequently modified by TET1 to 5hmC. The results suggest that the DNA repair enzyme ALKBH2 is important to reverse the etheno damage in ε5mC, and both ALKBH2 and ε5mC adduct may play wider roles in epigenetic modulation.

By investigating 1,N6-ethenoadenine (εA) repair across 16 possible sequence contexts (5’/3’ flanking base to εA varied as G/A/T/C), we revealed that repair efficiency is altered according to sequence, enzyme, and strand context (ss- versus ds-DNA). The results can be compiled into the repair aspect of εA mutation spectra. The same approaches can be used to study other pathways of repair or other aspects of mutational spectra including adduct formation and replication bypass.

In conclusion, this dissertation presents a methodical approach to carry out DNA adduct repair, bypass, and mutagenesis in vitro and in cell. The techniques used in this work can be used to study other pathways of repair as well as adding new dimensions to the study of DNA chemical biology.

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