Analysis of the effect of the G-QU adruplex fifth domain on DNA folding in response to oxideative and abasic lesions

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Publication Type dissertation
School or College College of Science
Department Chemistry
Author Omaga, Carla Alvarez
Title Analysis of the effect of the G-QU adruplex fifth domain on DNA folding in response to oxideative and abasic lesions
Date 2018
Description Damage to DNA due to endogenous and exogenous oxidants must be reversed by the cell through a complex system of DNA repair processes. At the forefront of this cellular defense mechanism is the DNA glycosylase NEIL3. This enzyme is encoded by the NEIL3 gene that possesses a G-rich promoter that has the potential to fold into a G-quadruplex (G4) structure. In this work, biophysical and biochemical methods (i.e., nuclear magnetic resonance spectroscopy, circular dichroism, thermal stability analysis, fluorescent probe detection, DNA polymerase stop assays) were used to demonstrate that the human NEIL3 promoter sequence adopts a dynamic mixture of G4 folds under near physiological conditions. Under an oxidative stress context, the properties of the NEIL3 G4 were altered in response to the presence of 8-oxo-7,8-dihydroguanine (OG) and required the fifth domain to stabilize DNA folding. The ability of the fifth domain to function as a "spare tire" by looping out the damaged G-track and reestablishing the G4 fold is the key feature that enabled the NEIL3 G4 to block the progression of DNA polymerase I (Klenow Fragment). However, compared to well-known oncogene G4s, the NEIL3 G4 was more readily bypassed by the DNA polymerase due to its highly dynamic polymorphism. The magnesium cation (Mg2+) is another factor that was discovered to enhance the stability of the NEIL3 G4 in the presence of oxidative damage. When OG is incorporated, the efficiency of the NEIL3 G4 to block the DNA polymerase is increased with the help of Mg2+ in a concentration-dependent manner. The findings gathered in this study underline the connection between magnesium and the formation of the NEIL3 G4 under oxidative conditions. The occurrence and effect of an abasic site in a G4 was also addressed in this work using the VEGF promoter G4 as a model sequence. The results showed that the abasic site destabilizes the VEGF G4, but the thermal stability and efficiency to block the DNA polymerase is restored when the fifth G-run served as a "spare tire" domain. The topology of the VEGF G4 was also changed by the presence of the fifth domain, a sequence-specific event that was not observed in the NEIL3 G4. Moreover, the folded structure of the VEGF G4 rendered it inaccessible for the apurinic/apyrimidinic endonuclease 1 (APE1) to bind and cleave the abasic site. However, the fifth G-run was revealed to facilitate the abasic site recognition and removal by APE1. These observations reveal that the fifth domain in G4-forming sequences adapts to a range of DNA lesions and may perform an essential role in DNA repair in vivo.
Type Text
Publisher University of Utah
Dissertation Name Doctor of Philosophy
Language eng
Rights Management (c) Carla Alvarez Omaga
Format Medium application/pdf
ARK ark:/87278/s6839rjq
Setname ir_etd
ID 1699893
Reference URL https://collections.lib.utah.edu/ark:/87278/s6839rjq
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