Structure and Function of DNA Repair Enzyme and Their Homologous Proteins

我们的目的是主要通过测定其三维结构来阐明DNA修复酶如光解酶的同源蛋白的结构与功能的关系。隐花色素是一种以黄素分子为修复基团的蓝光受体，其氨基酸序列与光解酶有很高的同源性，但不具有DNA修复活性，控制着动物的昼夜节律。CRY有一个FAD分子和第二个生色团作为光感受器的修复基团。我们构建了果蝇和水稻Crys的表达系统，并对重组蛋白进行了纯化。此外，我们还表达并纯化了嗜热古生菌tokodaii的光解酶重组蛋白和三趾鱼CPD(环丁烷嘧啶二聚体)类光解酶的重组蛋白。对纯化的重组蛋白进行了表征和定向结晶，并进行了X射线结晶学分析。在这些靶蛋白中，我们发现了…首次在2.8Å分辨率下测定了木香光解酶的晶体结构，这是古生菌光解酶三维结构的第一例。在这个光解酶分子中发现了两个FAD分子，其中FAD不仅作为催化辅因子结合到通常的FAD结合部位，而且还结合到捕光辅因子的结合部位。对于蓝藻光解酶，我们测定了脱辅蛋白状态(不含捕光辅因子8-HDF)的晶体结构，并研究了催化辅因子FAD的还原对蛋白质分子结构变化的影响。此外，作为含有FAD分子的蓝光受体蛋白CREY的功能同源蛋白，我们在2Å分辨率下测定了嗜热蓝藻细长热球菌BP-1的BLUF结构域的晶体结构。在晶体结构的基础上，我们讨论了Gln50与关键的Tyr8(Fad-Gln50-Tyr8网络)在结构和功能上与BLUF蛋白的光诱导光谱漂移有关的可能作用。较少

We aimed to elucidate the structure-function relationship of homologous proteins to DNA repair enzymes such as photolyase mainly by means of determination of their three-dimensional structures. Cryptochrome (CRY), one of blue-light receptors containing a flavin molecule as a prosthetic group, which has high homology in amino acid sequences with photolyase but no DNA repairing activity, controls the animal circadian rhythm. CRY has a FAD molecule and a second chromophore as prosthetic groups for light receptor. We constructed the expression system for CRYs from Drosophila melanogaster and Oryza sativa and purified recombinant proteins. We also expressed and purified the recombinant proteins for photolyase from a thermophilic archaea, Sulfolobus tokodaii and Class II CPD (cyclobutane pyrimidine dimer) photolyase from Potorous tridactylis. Purified recombinant proteins were characterized and targeted for crystallization to perform X-ray crystallography. Among these target proteins, we suc … More ceeded in crystal structure determination of photolyase from Sulfolobus tokodaii at 2.8Å resolution as the first case of the three-dimensional structure of archaeal photolyases. Two FAD molecules were found in this photolyase molecule where FAD is bound not only to the usual FAD binding site as a catalytic cofactor but also to the binding site for the light-harvesting cofactor. For cyanobacterial photolyase from Anacyctis nidulans, we determined crystal structures of an apoprotein state (without its light-harvesting cofactor, 8-HDF) and investigated how reduction of the catalytic cofactor, FAD affects on structural changes of the protein molecule. In addition, as a functionally homologous protein to CRY that is a blue-light receptor containing a FAD molecule, we determined the crystal structure of a BLUF domain from a thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1 at 2Å resolution. On the basis of the crystal structure, we discussed a possible role of Gln50,which is structurally and functionally linked with the critical Tyr8 (FAD-Gln50-Tyr8 network), with regard to the light-induced spectral shift of the BLUF proteins. Less