原核（6-4）DNA光修复酶和（6-4）光产物复合物晶体结构研究

Photolyases can repair ultraviolet (UV) light induced DNA damage with high efficiency and specificity to maintain the genetic integrity and accuracy of organisms thus preventing mutagenesis and cancer. PhrB from Agrobacterium fabrumis is the first prokaryotic (6-4) photolyase. Former structural and biochemical studies on PhrB have suggested significant differences between prokaryotic (6-4) photolyases and eukaryotic (6-4) photolyases. However，direct evidences are still missing for many details on DNA binding, electron transferring and energy transmitting processes. Here we would like to co-crystalize PhrB protein with single/double-stranded DNA (6-4) photoproducts and determine their crystal structures. The substrate binding mode of prokaryotic (6-4) photolyases will be established and the critical amino acids or motifs would be verified through combined site-directed mutagenesis with biochemical and functional studies. The elucidation of prokaryotic (6-4) photolyases functional mechanism can provide a molecular basis for exploring genetically modified organisms with enhanced UV-resistance for energy and food production.

DNA光修复酶能够对紫外线造成的DNA损伤进行高效特异性修复，维护遗传物质的完整性和准确性，避免由其引发的突变或癌变。根癌农杆菌（Agrobacterium fabrum）PhrB是首个被鉴定的原核（6-4）DNA光修复酶。前期关于PhrB蛋白质晶体结构和催化特性的研究表明，原核（6-4）DNA光修复酶与真核（6-4）DNA光修复酶之间存在显著差异，但在DNA结合、电子与能量传递等重要细节方面仍缺乏直接证据。本项目拟通过蛋白质晶体学技术获得首个原核（6-4）DNA光修复酶与单链/双链DNA底物复合物晶体结构，直接观测DNA结合模式，并通过构建突变体验证基于晶体结构提出的关键假说，揭示原核（6-4）DNA光修复酶与真核（6-4）DNA光修复酶在DNA结合与修复机制方面的异同。深入理解原核（6-4）DNA光修复酶的催化机理可为通过基因工程增强相关物种抗紫外线能力促进能源和食物生产提供科学基础。

DNA光修复酶能够对紫外线造成的DNA损伤进行高效特异性修复，维护遗传物质的完整性和准确性，避免由其引发的突变或癌变。根癌农杆菌（Agrobacterium fabrum）PhrB是首个被鉴定的原核（6-4）DNA光修复酶。前期关于PhrB蛋白质晶体结构和催化特性的研究表明，原核（6-4）DNA光修复酶与真核（6-4）DNA光修复酶之间存在显著差异，但在DNA结合、电子与能量传递等重要细节方面仍缺乏直接证据。本项目通过结构生物学技术进行了原核（6-4）DNA光修复酶与单链/双链DNA底物复合物的共结晶研究。表达纯化了野生型和三个突变体蛋白质并对其生物化学特性和体外修复功能和修复效率进行了检测。通过紫外线照射制备了四种含有光损伤位点的DNA底物并利用HPLC进行了大规模纯化。构建了原核（6-4）DNA光修复酶与DNA结合的三维结构模型。所获结果为深入理解原核（6-4）DNA光修复酶的催化机理进而通过基因工程增强相关物种抗紫外线能力促进能源和食物生产提供科学基础。