RUI: Toward a Molecular Understanding of DNA-Protein Crosslinking via Guanine Oxidation

Mount Saint Mary'S University, Los Angeles CA

Investigators

Abstract

Oxidative damage to DNA is a major stress in a living system. This project will study the mechanisms of oxidative damage using the flash-quench technique, which selectively oxidizes only the guanine base in DNA. In this photochemical method, a DNA-binding molecule is excited by light (the flash) and then donates an electron to an electron acceptor (the quench); the resulting oxidant then removes an electron from guanine to produce a reactive guanine radical, which in turn can form a permanent bond with protein (a crosslink). The overall objective of this work is to understand the molecular details of how the removal of an electron from guanine leads to DNA-protein crosslinks, and there are several specific goals for this research. First, the effect of local DNA environment, thought to alter the oxidation potential of guanine and the reactivity of guanine radical, will be examined via gel electrophoresis and spectroscopic methods. Next, the relative importance of crosslinking as a reaction pathway will be assessed by using HPLC to compare the amount of crosslinked guanines to the amount of other oxidized guanine species. Lastly, because there is a paucity of structural information regarding DNA-protein crosslinks, specific crosslinks will be produced in high enough yield for characterization. This approach will initially involve model systems where only one type of amino acid-base adduct is expected, and progress to the formation of specific crosslinks by utilizing DNA sequences with sites particularly vulnerable to oxidation. Enzymatic digestion, followed by HPLC purification, will be employed to isolate the crosslinked moiety, which will then be characterized by mass spectrometry, UV-visible spectroscopy and NMR spectroscopy. Broader impact. This research at the chemistry-biology interface will provide a significant training opportunity for undergraduates, as the multidisciplinary approach will require students to learn a wide variety of biochemical methods and spectroscopic techniques. In addition, this project should contribute to the advancement of underrepresented groups in science, since the student population at the host institution is primarily female and reflects the rich ethnic diversity of Los Angeles.