Campus Access Only
All rights reserved. This publication is intended for use solely by faculty, students, and staff of University of the Pacific. No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, now known or later developed, including but not limited to photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the author or the publisher.
Date of Award
2014
Document Type
Dissertation - Pacific Access Restricted
Degree Name
Doctor of Philosophy (Ph.D.)
Department
Pharmaceutical and Chemical Sciences
First Advisor
Liang Xue
First Committee Member
Andreas Franz
Second Committee Member
Wade Russu
Third Committee Member
Vyacheslav Samoshin
Fourth Committee Member
Jerry Tsai
Abstract
Exposure of DNA to reactive oxygen species (ROS) results in the modified nucleobases (lesions) as well as strand scissions under physiological conditions. Due to its lowest oxidation potential (1.29 eV), guanine is the most easily oxidisable nucleobase. Furthermore, it has been observed that the 5'-guanine in G-tracts (e.g. GGG) has even lower oxidation potential (1.00 V vs. NHE). One of the representative G-rich examples is telomeres that consist of repeating units of 5'-d [TTAGGG]-3' found at the ends of chromosomes. Telomeres play an important role in biological functions, serving as guardians of genome stability; however, their G-rich nature implies that they can be readily oxidized. So how does nature protect these biologically important regions from oxidation? We believe the formation of a secondary structure known as G-Quadruplex in telomeric regions can partly serve as a protective role. In the first part of this work, we investigated DNA G-Quadruplex damage under various oxidation conditions and compare the damage results with single-stranded telomeric sequences. Damage to G-Quadruplex is generally less than single strands and is condition dependent. Guanines are the primary damage sites, but damage of adenine and thymine is also possible. Based on our studies, telomeric DNA can be readily oxidized to produce DNA lesions. How do DNA lesions affect the conformation and the stability of telomeric G-Quadruplex DNA? In the second part, we sought to address this question using various biophysical methods. Several native (OxodG, OxodA, and abasic site) and non-native (8-NH 2 -dA and 8-Br-dA) lesions were tested. UV thermal denaturation and circular dichroism revealed that the conformation and the stability of G-Quadruplex DNA are dependent on the location and the type of lesion in the sequence. G-Quadruplex DNA containing OxodG maintains its conformation with a decreased stability. Abasic site in the TTA loop affects the conformation of G-Quadruplex DNA but shows little effect on its stability. An unexpected stabilization of telomeric G-Quadruplex DNA was observed when deoxyadenosine (dA) in the loops was replaced with its native oxidized form OxodA. This is the first example of native DNA lesion that increases the stability of G-Quadruplex DNA. Like OxodA lesion, 8-NH 2 -dA (a non native DNA lesion) increases the stability of G-Quadruplex DNA while 8-Br-dA only affects the stability in KCl but has no significant effect in NaCl. In addition, studies of the effect of OxodA lesion on the human telomerase activity using TRAP assay will be discussed.
Pages
242
ISBN
9781303681608
Recommended Citation
Aggrawal, Manali. (2014). Study of DNA damage on DNA G-quadruplexes and biophysical evaluation of the effects of modified bases (lesions) on their conformation and stability. University of the Pacific, Dissertation - Pacific Access Restricted. https://scholarlycommons.pacific.edu/uop_etds/134
Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.
To access this thesis/dissertation you must have a valid pacific.edu email address and log-in to Scholarly Commons.
Find in ProQuestIf you are the author and would like to grant permission to make your work openly accessible, please email
Rights Statement
In Copyright. URI: http://rightsstatements.org/vocab/InC/1.0/
This Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s).