Title Information
Name: Personal
Name: Personal
Name: Personal
Name: Personal
Name: Personal
Name: Personal
Name: Corporate
Origin Information
Physical Description
Note: thesis: Thesis (Ph. D.)--Brown University, 2019
Genre (aat): theses
Abstract: Despite being essential for organismal survival, genomic DNA is chemically reactive and susceptible to modification to generate mutagenic and/or cytotoxic lesions. These lesions include oxidation, alkylation, and deamination of the nucleobases. The removal of modified nucleobase lesions is the responsibility of the base excision repair (BER) pathway. BER is initiated by glycosylases that search for and remove the lesions. Previous characterizations of glycosylase activity have used DNA oligonucleotides, but a majority of eukaryotic DNA exists in a packaged environment. The basic unit of DNA packaging is the nucleosome core particle (NCP). This thesis focuses on characterizing the activity of three representative glycosylase-lesion pairs in NCPs: (1) human oxoguanine DNA glycosylase (hOGG1) on 8-oxo-7,8-dihydroguanine, (2) uracil DNA glycosylase (UDG) on uracil (U), and (3) human alkyladenine DNA glycosylase (AAG) on 1,N6-ethenoadenine (εA). hOGG1 and UDG activity was evaluated using kinetic studies of lesions positioned in various rotational positions near the DNA ends of the NCP. Lesion repair was enhanced relative to analogous rotational positions located on the dyad, suggesting that transient unwrapping of DNA ends facilitated repair. Additionally, histone tails were shown to suppress hOGG1 activity, but not UDG, emphasizing that local NCP architectural features can differentially influence glycosylase activity. UDG searching for lesions was also characterized in NCPs, specifically its processivity, or ability to remove multiple U in a single DNA binding event. Preliminary results show UDG processivity is greatly reduced in NCPs relative to duplex. The small amount of processivity observed is due to dissociative transfer (“hopping”) with no contribution of associative transfer (“sliding”). Lastly, AAG activity was characterized across a variety of translational and rotational positions simultaneously utilizing a global εA-containing NCP population to determine kinetic parameters at 49 distinct sites. Overall, the amount of excision by AAG correlated with solution accessibility characterized by hydroxyl radical footprinting and exhibited monophasic kinetics. Exceptions to this repair-accessibility correlation are likely due to local NCP architectures, such as differences in DNA dynamics, DNA wrapping, and interaction with histone tails. These in vitro experiments provide a molecular understanding for biological observations between DNA damage, repair, and mutation in human disease.
Subject (fast) (authorityURI="http://id.worldcat.org/fast", valueURI="http://id.worldcat.org/fast/01024734")
Subject (fast) (authorityURI="http://id.worldcat.org/fast", valueURI="http://id.worldcat.org/fast/00859922")
Subject (fast) (authorityURI="http://id.worldcat.org/fast", valueURI="http://id.worldcat.org/fast/00831961")
Subject (fast) (authorityURI="http://id.worldcat.org/fast", valueURI="http://id.worldcat.org/fast/00886599")
Subject (fast) (authorityURI="http://id.worldcat.org/fast", valueURI="http://id.worldcat.org/fast/00913605")
Subject
Subject (fast) (authorityURI="http://id.worldcat.org/fast", valueURI="http://id.worldcat.org/fast/00886581")
Language
Record Information
Identifier: DOI: 10.26300/8nt8-2266
Access Condition: rights statement (href="http://rightsstatements.org/vocab/InC/1.0/"): In Copyright
Access Condition: restriction on access: All rights reserved. Collection is open to the Brown community for research.
Type of Resource (primo): dissertations