Characterization of Tryptophan Radical and Flavin Excited State Intermediates in DNA Photolyase Electron Transfer Reactions

DNA特定的损伤是由于DNA暴露在紫外线下造成的。主要的光产物是由于相邻的核酸碱基的二聚化，即环丁烷嘧啶二聚体(CPD)。许多生物体使用一种非常有效的DNA修复酶--DNA光解酶来逆转这种DNA特有的损伤。DNA光解酶属于蓝光感受器的一类，它通过光驱动的电子转移机制逆转突变的CPD损伤。当酶是从其活性形式氧化的一个电子时，它可以进行光还原反应返回到活性形式，这可能涉及通过色氨酸氨基酸链进行电子转移，这些氨基酸在蓝光感受器类中是高度保守的。在这个项目中，将使用最先进的超快激光光谱来确定光吸收后光解酶活性部位的结构变化，以了解DNA光修复的主要过程，以及损伤DNA的存在如何在分子水平上改变这些过程。同样的方法将被用来表征参与色氨酸电子转移过程的个别反应中间体。该方法使用具有皮秒时间分辨率(百万分之一秒)的时间分辨共振拉曼光谱，黄素分子和色氨酸残基的生物合成同位素标记，以及高级计算来解释结果。该项目还包括培训研究生和博士后研究员应用超快激光光谱学，以及培训本科生使用的基本生物物理、生物化学和光谱学方法。

DNA-specific damage results from exposure of DNA to ultraviolet light. The major photoproduct is due to dimerization of adjacent nucleic acid bases, the cyclobutane pyrimidine dimer (CPD). Many organisms use a very efficient DNA repair enzyme, DNA photolyase, to reverse this DNA-specific damage. DNA photolyase belongs to the class of blue-light photoreceptors and reverses the mutagenic CPD damage via a light-driven, electron transfer mechanism. When the enzyme is one electron oxidized from its active form, it can undergo a photoreduction reaction back to the active form which presumably involves electron transfer through a chain of tryptophan amino acids which are highly conserved within the class of blue-light photoreceptors. In this project, state-of-the-art ultrafast laser spectroscopy will be used to determine the structural changes in the photolyase active site after absorption of light to understand the primary processes of DNA photorepair and how the presence of damaged DNA modifies these processes on a molecular level. The same methodology will be applied to characterize the individual reaction intermediates that are involved in the tryptophan electron transfer process. The approach uses time-resolved resonance Raman spectroscopy with picosecond time-resolution (one millionth of a millionth of a second), biosynthetic isotopic labeling of the flavin molecule and tryptophan residues, and high-level calculations to interpret the results. The project also involves the training of graduate students and postdoctoral research associates in the application of ultrafast laser spectroscopy, along with training of undergraduate students in the basic biophysics, biochemistry and spectroscopy methods used.