A System for Comparing Relative Rates of Back Electron Transfer and Crosslinking
Authors:
Christina Khorozyan, Shushan Khorozyan, Melissa Marquez, Kelsey Miller, Zitadel Anne PerezMentor:
Eric Stemp, Department Chair, Physical Sciences, Mount St. Mary's CollegeOxidative damage to DNA is important in the progression of molecular diseases such as cancer. Electron holes produced upon DNA oxidation are mobile and can move throughout the base stack. One possible trapping reaction for guanine radicals, produced upon oxidation, is the formation of DNA-protein crosslinks. The fluorescent base analogue, 2-aminopurine (Ap) is capable of photo-oxidizing guanine but also undergoes back electron transfer with the guanine radical produced. We placed 2-aminopurine into a DNA strand at two different distances from the nearest guanine base to determine the relative rates of back electron transfer and crosslinking to histone protein. Crosslinking was detected by the gel shift assay. A 29-mer DNA duplex containing a 5’- G(T)nX -3’ sequence was used, where n=0 or 4 and X= 2-aminopurine. Upon irradiation, no change was seen in the mobility of the n=0 duplex. In contrast, the n=4 duplex showed a decrease in free DNA bands and the appearance in bands of lower mobility, consistent with crosslinking for this DNA. One explanation for these results is that in the n=4 duplex, back electron transfer, due to the longer distance, does not compete as well with the trapping reaction with protein.