Photoinduced electron transfer in DNA photolyase

Temple University, Philadelphia PA

Investigators

Abstract

This award funded by the Experimental Physical Chemistry Program of the Chemistry Division supports research by Professor Robert Stanley from Temple University to study electron transfer-mediated repair of DNA. DNA is an extraordinarily stable molecule, a property required in its role as a genetic storage medium. In spite of this stability, DNA is constantly being damaged by a variety of environmental agents. Of these, ultraviolet (UV) light is among the most mutagenic, leading to a crosslinking of adjacent pyrimidines to generate Cyclobutylpyrimidine Dimers (CPDs). All organisms have a robust ability to repair CPDs. DNA photolyase (PL) is a protein that binds and repairs CPDs with exquisite specificity. Uniquely, photolyase uses visible light as an energy source. Once bound, the CPD is repaired in about two billionths of a second after the absorption of the catalytic photon. This photon is absorbed by the protein-bound Flavin Adenine Dinucleotide (FAD), a vitamin B2 derivative which transfers an electron to the CPD. This critical ultrafast electron transfer step is not well understood. Professor Stanley and his students will use state-of-the-art biophysical, biochemical, and molecular biology methods to determine the details of this electron transfer mechanism at the molecular level. In particular, fluorescence Stark spectroscopy will be utilized for the first time in the study of flavoproteins to discover the direction of the electron transfer, thereby revealing the identity of the initial electron acceptor. Mutants will be made to elucidate the role of amino acids around the flavin in the repair reaction. This approach, coupled with an analysis of the excited state electronic structure obtained using Stark spectroscopy will provide the clearest picture of how and why photolyase functions. More broadly, this application of Stark spectroscopy to PL will be directly applicable to other light-driven flavoproteins (e.g. blue light photoreceptors) which utilize different photochemical mechanisms but which, like PL, begin with light-driven charge redistribution in the flavin. Some of these proteins may be responsible for regulating our circadian clock or providing birds with the ability to migrate using the Earth's magnetic field for guidance (photomagnetoreception). Still other proteins are known to regulate photosynthesis at the gene level. Two collaborators join this effort. Professor David Beratan of Duke University will provide computational models to guide the interpretation of the experiments. Professor Yvonne Gindt and her group of undergraduate researchers at Lafayette College will perform electrochemical measurements on modified proteins to gauge the effect of chemical and mutational changes on the redox properties of the FAD. Postdoctoral and graduate students will gain a breadth of experience that is a hallmark of biophysical chemistry, learning techniques that include Stark and subpicosecond ultrafast laser spectroscopy, enzymology, molecular biology, and modern computational methods. Important experiments will be performed by a cadre of talented undergraduate students both at Temple and at Lafayette College in Easton, PA. These budding scientists will gain research experience by purifying the photolyase protein and performing chemical and molecular biological modifications to probe the true function of the FAD. All participants will present their results twice a year in a "mini-conference" setting, to take place alternately in Philadelphia and Easton and involving the Duke group through video-conferencing. The PI has mentored many young scientists, including women and members of underrepresented minorities. To reach a non-scientific audience about the importance of science to society, the PI has been closely involved in the TURF-Crews program at Temple for several years. This program mixes presentations by undergraduates from non-scientific fields with those by science majors to engender multidisciplinary interactions.