DNA Lesions Produced from the Reaction of Diols and 5-Formylcytosine and Their Effects on DNA Replication

Author

Brock Allen, University of the PacificFollow

Date of Award

2018

Document Type

Thesis

Degree Name

Master of Science (M.S.)

Department

Pharmaceutical and Chemical Sciences

First Advisor

Liang Xue

First Committee Member

Jerry Tsai

Second Committee Member

Lisa A. Wrischnik

Abstract

Nucleic acids are complex macromolecules that are susceptible to both endogenous and exogenous damage. This study explored damage resulting from interactions with environmental nucleophilic toxins, such as a variety of diols and amines found in industry. These nucleophiles can react with electrophilic groups, such as 5-formylcytosine. 5-formylcytosine is an oxidation product of the epigenetic base 5-methylcytosine. It is typically removed by thymine DNA glycosylase (TDG) but is known to accumulate in the genome, making the formyl group susceptible to attack. In this study we used GC/MS and ESI-MS to show that DNA lesions from the nucleophilic addition reaction of toxins and 5-formylcytosine can be formed under physiological conditions. In addition, this formation showed a pH dependency, with lower pHs showing more product formation. Studies with a lesion formed from the reaction of 1,3-propane diol and 5-formylcytosine showed that the lesion has little effect on the conformation of the DNA duplex. UV thermal denaturation studies showed that at a glance the lesion also has little effect on the stability of the DNA duplex, however, more extensive studies revealed a slight destabilization effect due to the lesion. Enzymatic studies showed that the presence of one lesion does not have a significant effect on the ability of DNA polymerase to efficiently complete DNA replication with high fidelity, but when the lesion was incorrectly base paired, the extension reactions resulted in deletion products or a halt in replication. Addition of a second tandem lesion to the template resulted in a decrease in fidelity, while continuing to give deletion products and replication stops in the presence of mismatched base pairs. This is particularly significant, indicating the potential for the lesion to be mutagenic or even cytotoxic. Lesions formed from other environmental toxins could be even more damaging, making them well worth future investigation.

Pages

132

Recommended Citation

Allen, Brock. (2018). DNA Lesions Produced from the Reaction of Diols and 5-Formylcytosine and Their Effects on DNA Replication. University of the Pacific, Thesis. https://scholarlycommons.pacific.edu/uop_etds/3539