MCB 9817338 1. Technical: A new method is proposed for the generation of covalent bonds between DNA and protein (DNA-protein crosslinks). These covalent adducts are a potentially lethal form of DNA damage, and can be formed from attack of proteins on oxidatively damaged DNA. Using a photochemical method called the flash-quench technique, a powerful ground-state oxidant will be prepared on the DNA by oxidative quenching of a photoexcited intercalator. The oxidized intercalator selectively oxidizes guanine bases. Given that DNA is typically associated with proteins in the cell, and that proteins have many nucleophilic sidechains, one likely outcome following guanine oxidation is the formation of DNA-protein crosslinks. Current experimental approaches to DNA-protein crosslinking reactions produce either a myriad of nonspecifically crosslinked products or specific products that are not of physiological relevance. In contrast, the flash-quench technique offers a way to produce specific crosslinks between DNA and protein that may have physiological relevance. The specific aims of this study are to i) define and optimize the parameters important for generating nonspecific DNA-protein adducts by the flash-quench technique and ii) create specific DNA-protein crosslinks to obtain one adduct in high yield for chemical characterization. The successful establishment of this approach would help to elucidate one consequence of oxidative damage to DNA at the molecular level. This research experience will be shared with the students.
2. Non-technical: A new method is proposed for the formation of strong bonds between DNA and protein, known as DNA-protein crosslinks. Oxidative damage to DNA is thought to play a role in many diseases, and many people take antioxidants to counteract the free radicals that can cause such damage. Free radicals are electron-deficient species that can oxidize (take electrons from) DNA, leaving an "electron hole" on the DNA. It is thought that this "electron hole" can be filled by amino acids of proteins that are electron-rich, resulting in a bond between DNA and protein. Because DNA must be free to interact with many different proteins in the body, the formation of a permanent bond with one given protein could be lethal. Here, the flash-quench technique is used to make electron holes on DNA. In this method, a "flash" of blue light excites a small molecule nestled between the DNA bases, and this excited molecule is then "quenched" by giving an electron to another small molecule nearby the DNA. The quenched molecule then behaves like a free radical and oxidizes the DNA, which in turn leads to DNA-protein crosslinks. The advantage of the flash-quench technique is that it only oxidizes one type of group on the DNA, the guanine base, whereas most other approaches heretofore cause indiscriminate damage all over the DNA molecule. This selectivity should allow for the detailed study of DNA-protein crosslinking reactions with guanine, and should elucidate one consequence of oxidative damage to DNA at the molecular level. This research experience will be shared with the students.
