Date of Award
2024
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 assaults 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. The first section focuses on investigations of the guanine adduct generated by Cinnamaldehyde (CA) and its mutagenesis, and the other section discusses how 2OG/Fe(II)-dependent dioxygenases, such as AlkB, repair DNA adducts.
By developing an organic solvent-based reaction, we are able to synthesize a DNA oligonucleotide containing a CA adduct, then we developed a Liquid Chromatography based method to purify the DNA with CA adducts. The results showed that CA can form two isomers with Guanine with a 1:1 ratio distribution.
By employing a mass spectrometry-based approach, we were able to evaluate the in vivo bypass and mutagenesis of CA adducts. The findings indicate that the two isomers exhibit comparable mutation patterns, with approximately 50% of the CA-G adducts being repaired back to G in cells. The most frequent mutation involves a G to C conversion, accounting for about 45% of the CA-G adduct mutations. Mutations to A and T are relatively rare, occurring in less than 1% of cases.
The second part (chapter 2 and 3) focuses on DNA repair mediated by AlkB. By examining the repair kinetics of AlkB enzymes for various nucleobase modifications and comparing their kinetic parameters with their repair efficiencies in vivo, we established a correlation between in vitro and in vivo repair mechanisms. We introduced a novel metric, the "final repair ratio," which incorporates both the bypass and mutation ratios. Our findings reveal a significant correlation between the in vitro catalytic efficiency (Kcat/Km) and the in vivo final repair ratio. Specifically, a higher Kcat/Km value corresponds to an increased final repair ratio. This relationship enables the prediction of in vivo repair efficiency based on in vitro Kcat/Km values, offering valuable insights for future research into the in vivo activity of AlkB family enzymes and their human homologs.
By analyzing the kinetic parameter (Kcat/Km) of AlkB with various substrates, encompassing both single-stranded and double-stranded forms, we conducted a comprehensive investigation into AlkB's substrate specificity and uncovered insights into its activity on single versus double strands. Additionally, we identified novel substrates for AlkB and assessed their activity by measuring their Kcat/Km values.
In conclusion, this dissertation introduces a systematic methodology for the synthesis, characterization, and purification of a DNA adduct, as well as for conducting in vitro and in-cell repair, bypass, and mutagenesis of DNA adducts. The techniques developed and applied in this study have broader applications, enabling the investigation of other chemicals that may form adducts and damage DNA. Furthermore, these methodologies can facilitate research into additional DNA repair pathways and contribute new perspectives to the field of DNA chemical biology.
Recommended Citation
Zhou, Xianhao, "DNA DAMAGE, MODIFICATION AND REPAIR" (2024). Open Access Dissertations. Paper 1656.
https://digitalcommons.uri.edu/oa_diss/1656