DNA光解酶高效修复作用机理的研究

项目名称： DNA光解酶高效修复作用机理的研究

项目编号： No.30870581

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 化学工业

项目作者： 宋钦华

作者单位： 中国科学技术大学

项目金额： 32万元

中文摘要： DNA光损伤的二种主要光产物可分别被CPD光解酶和(6-4)光解酶通过光复活修复，二者修复量子效率迥异。揭示CPD光解酶高效修复的作用机理，对于认识这一生命现象的本质有重要意义。本项目合成了二类模型化合物，共价连接的嘧啶二聚体-电子给体（黄素、咔唑和吲哚等）和共价连接的氧杂环丁烷-电子给体，分别模拟二类光解酶的光复活过程，结果显示：1)底物与电子给体间构象影响修复效率；2)CPDs与(6-4)二类模型化合物修复过程中的电子转移反应处于不同的Marcus区域；3)黄素-CPDs模型修复效率低是因为短的黄素激发态寿命和快速的不能导致修复的逆向电子转移。同时，运用基因突变工程，合成了多个DNA光解酶突变体，研究发现Asn378对于稳定中性自由基FADH实现高效修复至关重要。这些结果揭示了DNA光解酶高效修复的机理：底物与酶的结合是高效修复的前提，DNA光解酶能够利用特定的几何形状和静电相互作用来固定黄素辅酶，达到1)增加其激发态的寿命，使其与CPDs有足够的反应时间；2)稳定电荷分离中间体，使CPDs有足够裂环时间。二步高效反应促成了完美的DNA光复活作用。

中文关键词： DNA光修复；CPD光解酶；嘧啶二聚体；修复效率；作用机理

英文摘要： The two major lesions in DNA upon irradiation with UV light, the cyclobutane pyrimidine dimers (CPDs), which are only cis-syn forms in double-stranded DNA and an additional trans-syn forms in a much smaller extent for single-stranded DNA, and pyrimidine (6-4) pyrimidone photoproducts, are responsible for the harmful effects of UV on organisms, respectively. The two photolesions can be repaired through DNA photoreactivation catalyzed by CPD photolyases and (6-4) photolyases in very different quantum efficiencies to utilize the energy of sun light (300-500 nm), respectively. In this project, two classes of model compounds, covalently linked pyrimidine dimer-donor (indol, carbazol or flavin) and pyrimidine oxetane-donor, were empolyed to mimic two photoreacivations mediated by two photolyases, respectively. Based on model studies, we have obtained conclusions as follows: 1)The splitting quantum efficiency depends on the molecular conformation between the dimer and a donor; 2)Photosensitized splitting of thymine dimer or oxetane unit by a covalently N-linked carbazole via electron transfer in different Marcus regions; 3)The underlying mechanism for the low repair quantum yield of flavin-thymine dimer models is the short-lived excited flavin moiety and the fast dynamics of futile back electron transfer without repair. Meanwhile, we have concluded that the Asn378 residue of E. coli photolyase is crucial both for stabilizing the neutral flavin radical cofactor and for catalysis. In general, by investigation of this project, the repair mechanism of CPD photolyases with high efficiencies has been demonstrated. In CPD photolyase, the enzyme can utilize geometric restriction and electrostatic interactions to confine the flavin cofactor and lengthen its excited-state lifetime. This long-lived cofactor could exist long enough to react with CPDs to form a charge-separated intermediate and thus reach a high ET quantum yield. Moreover, the active site stabilizes charge-separated intermediate in photolyase and speeds up the ring splitting. Such modulation of the dynamics leaves enough time to cleave the ring, resulting in a high splitting efficiency. These two high efficient processes lead to a perfect repair of damage DNA by photolyase.

英文关键词： DNA photoreactivation; DNA photolyase; pyrimidine dimer; Repair efficiency; Mechanism