Oxidaton of Endonuclease III by Guanine Radicals
Authors:Anna Arnold, Jackie K. Barton, Shushan Khorozyan
Mentor:Eric D. A. Stemp, Department Chair, Physical Sciences , Mount St Mary's College
Oxidative damage to DNA is important in the progression of molecular diseases such as cancer and guanine is particularly susceptible due to its low oxidation potential. In vivo, oxidative lesions are often corrected by base excision repair (BER) enzymes such as MutY and it has been proposed that since many DNA glycosylases have FeS clusters that are redox-active once the protein binds to DNA, these proteins might use through-DNA charge transfer (CT) to scan the DNA for defects. Oxidation of guanine leads to MutY oxidation and our goal was to demonstrate that EndonucleaseIII can exhibit similar behavior. Here, we designed oligonucleotides containing 2-aminopurine (a fluorescent base that can photooxidize guanine) and a series of guanines decreasing in ionization potential with increasing distance from the 2-aminopurine, which should helping to minimize back electron transfer. In a model protein, we observed oxidation of ferrocytochrome c upon irradiation of the 2-aminopurine, consistent with protein-to-DNA electron transfer. With that, our future goal is to monitor reaction between guanine radical and Endo III in real time using nanosecond transient absorption spectroscopy and obtain evidence for permanent oxidation products by gel electrophoresis and uv-visible spectroscopy.