Dynamics and Mechanism of DNA-Repair Photolyase and Circadian Cryptochrome

描述(申请人提供)：光解酶是一种光酶，它利用蓝光的能量来逆转许多生物体中紫外线诱导的DNA损伤。隐花色素是最近发现的一种蓝光感受器，它调节动物(和植物)的生物钟以及植物的生长发育。这两种蛋白质的结构相似，但功能完全不同。我们最近已经阐明了一类微生物光解酶(Class-I)对环丁烷嘧啶二聚体的修复机制和光循环，以及(6-4)光解酶对嘧啶-嘧啶(6-4)光解产物的修复机制和光循环。最近发现了几类新的具有新活性部位的光解酶，并观察到一些新的功能。因此，该项目的第一个目标(目标1)是系统地表征所有其他三类光解酶对环丁烷嘧啶二聚体的修复以及(6-4)光解酶对杜瓦修复的新功能。这样的系统研究将获得对紫外线辐射对生物圈有害影响的分子理解。分子遗传学已经对隐花色素进行了大量的研究，但对其机理的研究和了解还很少。甚至在隐色素中辅因子黄素的活性(功能)氧化还原状态也是未知的。目前只知道隐花色素在蓝光接收时会发生构象变化，从而触发下游信号转导。因此，第二个目标(目标2)是系统地研究氧化还原状态(S)和相关的光化学，确定植物和动物隐色素在体内的活性状态，并表征随后的构象动力学以及与下游蛋白质的相关相互作用。这些研究将揭示初始信号转导的主要过程，揭示隐花色素的反应机理和光循环。为了实现这些目标，我们集成了最先进的激光光谱学和生物化学/分子生物学，并以飞秒时间分辨率和单残基空间分辨率跟踪DNA修复和复杂过程的初始信号的整个功能进化。从这项工作中获得的关于光解酶和隐花色素的新知识对DNA修复和生物时钟领域具有重要意义，更重要的是，对于针对皮肤癌和精神障碍等一系列疾病的地毯设计的实际应用至关重要。

DESCRIPTION (provided by applicant): Photolyase is a photoenzyme that uses the energy of blue light to reverse UV-induced DNA damage in many organisms. Cryptochrome is a recently discovered blue-light photoreceptor that regulates the circadian clock in animals (and plants) and growth and development in plants. Both proteins have similar structural architectures but with totally different functions. We have recently elucidated the repair mechanisms and photocycles of cyclobutane pyrimidine dimer by a class of microbial photolyases (class-I) and of pyrimidine-pyrimidone (6-4) photoproduct by (6-4) photolyases. Several new classes of photolyases have been recently discovered with novel active sites and some new functions have also been lately observed. Thus, the first objective (Aim 1) of the project is to systematically characterize the repair of cyclobutane pyrimidine dimer by all other three classes of photolyases and the new function of Dewar repair by (6-4) photolyases. Such systematic investigations will obtain the molecular understanding of detrimental effects of UV radiation on the biosphere. Cryptochrome has been heavily studied by molecular genetics but the mechanistic investigation and understanding are simply lacking. Even the active (functional) redox state of cofactor flavin in cryptochrome is unknown yet. It is only known that cryptochrome proceeds to conformational changes to trigger downstream signal transduction upon blue-light photoreception. Thus, the second objective (Aim 2) is to systematically investigate the redox state(s) and related photochemistry, determine the active state in vivo in plant and animal cryptochromes, and characterize subsequent conformation dynamics and related interactions with downstream proteins. These investigations will uncover the primary process of initial signal transduction and reveal the reaction mechanism and photocycle of cryptochrome. To achieve these goals, we integrate state-of-the-art laser spectroscopy and biochemistry/molecular biology and follow the entire functional evolution of DNA repair and initial signaling of the complex processes with femtosecond temporal resolution and single-residue spatial resolution. The new knowledge obtained from this work on photolyase and cryptochrome is significant to the DNA repair and biological clock fields and, more importantly, is critical to practical applications of rug design for a series of diseases such as skin cancer and mental disorder.