Functional Dynamics and Molecular Mechanism of Photolayse

说明(申请人提供)：光解酶的功能动力学和分子机制光解酶是一种光酶，它利用蓝光的能量来逆转许多生物体中紫外线诱导的DMA损伤。它在人类中是缺乏的，但最近在人类细胞系和小鼠身上的实验表明，通过基因治疗，光解酶可以用来提高对阳光诱导的肿瘤形成的抵抗力。环丁烷嘧啶二聚体(CPD)和嘧啶-嘧啶酮(6-4)光产物是紫外光照射细胞产生的两大类具有细胞毒性、致突变性和致癌性的DNA光产物，是引起皮肤癌的主要原因。光解酶修复DNA的假想分子模型已经被提出并研究了很长时间，但直到去年我们用超快光谱捕捉到催化中间体时，才观察到催化途径的直接证据。对于原子尺度上非常有效的修复机制，人们一直缺乏了解。因此，本项目的第一个目标是从最基本的水平上揭示CPD光解酶修复DNA二聚体的分子机制，并确定其高修复效率的关键因素。(6-4)光解酶修复(6-4)-光产物的机理研究较少，修复动力学也从未被研究过，其效率低于CPD光解酶。因此，第二个目标是表征(6-4)光解酶的修复动力学，阐明其分子机制，并了解导致其效率低于CPD光解酶的因素。为了实现这些目标，我们集成了最先进的飞秒激光光谱学、分子生物学方法和计算研究，并将修复过程的动态复杂性分解为基本步骤。利用飞秒时间分辨率和单残基空间分辨率，我们将揭开功能动力学的完整演化，揭示光解酶修复DNA的基本机制。通过这项工作获得的认识将有助于合理的药物设计和基因治疗，以预防紫外线诱发的皮肤癌。

DESCRIPTION (provided by applicant): Functional Dynamics and Molecular Mechanism of Photolyase Photolyase is a photoenzyme that uses the energy of blue light to reverse UV-induced DMA damage in many organisms. It is lacking in humans, but recent experiments in human cell lines and in mice indicate that through gene therapy, photolyase can be used to heighten resistence to sunlight-induced tumorigenesis. Cyclobutane pyrimidine dimer (CPD) and pyrimidine-pyrimidone (6-4) photoproduct are the two major classes of cytotoxic, mutagenic and carcinogenic DNA photoproducts produced by UV-light irradiation of cells and are the main cause of skin cancer. A hypothetical molecular model of DNA repair by photolyase has been proposed and investigated for a long time, but direct evidence for the catalytic pathway had never been observed until last year when we captured a catalytic intemediate using ultrafast spectroscopy. Understanding of the remarkably efficient repair mechanism at the atomic scale has been lacking. Thus, the first objective of the project is to reveal the molecular mechanism of dimer repair in DNA by CPD photolyase at the most fundamental level and determine the key factors for its high repair efficiency. The mechanism of the (6-4)-photoproduct repair by (6-4) photolyase has been less examined and the repair dynamics, less efficient than CPD photolyase, have never been explored. Therefore, the second objective is to characterize the repair dynamics of (6-4) photolyase, elucidate its molecular mechanism, and understand the factors contributing to its lessened efficiency compared to CPD photolyase. To achieve these goals, we integrate state-of-the-art femtosecond laser spectroscopy, molecular biology methods and computational studies, and break down the dynamic complexity of repair processes into elementary steps. With femtosecond temporal resolution and single-residue spatial resolution, we will unravel the complete evolution of the functional dynamics and lay bare the fundamental mechanisms of DNA repair by photolyase. The understanding gained from this work will help in rational drug design and gene therapy to prevent skin cancer induced by UV light.