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Publication Date

2023-5

First Advisor

Elizabeth R. Jamieson

Document Type

Honors Project

Degree Name

Bachelor of Arts

Department

Biochemistry

Keywords

Nucleosome, base excision repair, Spiroiminodihydantoin, DNA lesion, 8-oxoguanine, Formamidopyrimidine DNA glycosylase, Oxidative DNA damage, Ligation

Abstract

Deoxyribonucleic acid (DNA) is constantly under attack from reactive oxygen species (ROS) from endogenous and exogenous sources, which can cause various forms of damage, including mutagenic damage that threatens genomic integrity. Oxidative DNA damage has been implicated in inflammation, carcinogenesis, neurodegenerative diseases, and aging. Of the four canonical nucleobases, guanine (G) has the lowest reduction potential and is most easily oxidized to the lesion 8oxoguanine (8oxoG), which has an even lower reduction potential and can be further oxidized to hydantoin lesions. One of these lesions is spiroiminodihydantoin (Sp), a chiral propeller-shaped lesion that can destabilize the DNA duplex and transversion mutations. Oxidative lesions and other non-bulky lesions can be repaired by the base excision repair (BER) pathway, which occurs in five core steps. Bifunctional glycosylases can perform the first two of these steps: extrusion and excision of the damaged nucleobase, leaving an abasic (AP) site, which is then incised by AP lyase activity to form a single-strand break (SSB). The bacterial bifunctional glycosylase formamidopyrimidine (Fpg) can excise both 8oxoG and Sp at comparable rates in free duplex (leippold, krishnamurthy). However, most DNA in cells is packaged in nucleosomes: 145-147 base pairs of DNA wrapped around an octameric histone protein core. Nucleosomes form the polymer chromatin, which condenses into chromosomes and compacts the genome. Nucleosome core particles (NCPs) pose a steric hindrances to glycosylases and decrease the efficiency of excision. It is therefore necessary to study the kinetics of glycosylase activity in nucleosomes to better understand lesion excision in physiological conditions. This project’s eventual goal is to determine the rate of excision of Sp by Fpg in nucleosomes using an Fpg glycosylase assay. The extent of reaction can be visualized and quantified using polyacrylamide gel electrophoresis (PAGE). But in order to form nucleosomes, it is necessary to synthesize and purify strands of DNA long enough to wrap around a histone core.

Rights

©2023 Alina Jarrett. Access limited to the Smith College community and other researchers while on campus. Smith College community members also may access from off-campus using a Smith College log-in. Other off-campus researchers may request a copy through Interlibrary Loan for personal use.

Language

English

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