Dynamics and Mechanism of DNA-Repair Photolyase and Circadian Cryptochrome

DNA 修复光解酶和昼夜节律隐花色素的动力学和机制

• 批准号: 8838820
• 负责人: DONGPING ZHONG
• 金额: $ 28.12万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-02-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8838820
• 关键词: Active Sites; Address; Animals; Architecture; Binding; Biochemical; Biochemistry; Biological Clocks; C-terminal; Circadian Rhythms; Complex; DNA; DNA Damage; DNA Photolyase; DNA Repair; Disease; Distant; Drug Design; Energy-Generating Resources; Environment; Enzymes; Evolution; Flavins; Flavoproteins; Funding; Goals; Growth and Development function; Health; In Vitro; Investigation; Knowledge; Label; Lasers; Lead; Length; Light; Maps; Mental disorders; Methods; Molecular; Molecular Biology; Molecular Conformation; Molecular Genetics; Motion; Organism; Oxidation-Reduction; Photochemistry; Photoreceptors; Plants; Process; Proteins; Psyche structure; Pyrimidine; Pyrimidine Dimers; RNA; Reaction; Research; Resolution; Roentgen Rays; Series; Signal Transduction; Single-Stranded DNA; Site-Directed Mutagenesis; Skin Cancer; Spectrum Analysis; Structure; Sunlight; Time; UV induced DNA damage; Ultraviolet Rays; Work; absorption; circadian pacemaker; cofactor; cryptochrome; design; flavin semiquinone; in vivo; microbial; novel; plant growth/development; practical application; repaired; research study; semiquinone; signal processing; skin cancer prevention; temporal measurement; ultraviolet irradiation

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.

描述（由申请人提供）：光裂解酶是一种光酶，它利用蓝光的能量来逆转许多生物体中紫外线引起的 DNA 损伤。隐花色素是一种最近发现的蓝光光感受器，可调节动物（和植物）的生物钟以及植物的生长和发育。两种蛋白质具有相似的结构，但具有完全不同的功能。我们最近阐明了一类微生物光裂合酶（I 类）对环丁烷嘧啶二聚体的修复机制和光循环以及（6-4）光裂合酶对嘧啶-嘧啶酮（6-4）光产物的修复机制和光循环。最近发现了几类具有新活性位点的新光裂合酶，并且最近还观察到了一些新功能。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裂合酶对环丁烷嘧啶二聚体的修复以及（6-4）光裂合酶杜瓦修复的新功能。此类系统研究将从分子角度了解紫外线辐射对生物圈的有害影响。隐花色素已通过分子遗传学进行了大量研究，但缺乏机制研究和理解。甚至隐花色素中辅因子黄素的活性（功能）氧化还原状态也是未知的。只知道隐花色素在蓝光感光时发生构象变化以触发下游信号转导。因此，第二个目标（目标2）是系统地研究氧化还原态和相关的光化学，确定植物和动物隐花色素体内的活性状态，并表征随后的构象动力学和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，并揭示隐花色素的反应机制和光循环。为了实现这些目标，我们整合了最先进的激光光谱和生物化学/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传导的整个功能演化。这项关于光裂合酶和隐花色素的工作获得的新知识对于DNA修复和生物钟领域具有重要意义，更重要的是，对于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。