Analysis of the effect of the G-Quadruplex fifth domain on DNA folding in response to oxidative and abasic lesions

dissertationDamage 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