Structure and Function of DNA Repair Enzyme and Their Homologous Proteins

我们主要通过对光解酶等DNA修复酶的三维结构的测定来阐明其结构-功能关系。Cryptochrome （CRY）是一种以黄素分子为假基的蓝光受体，其氨基酸序列与光解酶高度同源，但不具有DNA修复活性，控制着动物的昼夜节律。CRY具有一个FAD分子和第二个发色团作为光受体的假体基团。我们构建了黑腹果蝇和水稻CRYs的表达体系，并纯化了重组蛋白。我们还表达并纯化了一种嗜热古菌、Sulfolobus tokodaii和Potorous tridactylis II类CPD（环丁烷嘧啶二聚体）光解酶重组蛋白。对纯化的重组蛋白进行了表征，并进行了x射线晶体学的结晶。在这些靶蛋白中，我们首先在2.8Å分辨率下测定了tokodaii Sulfolobus光解酶的晶体结构，这是古细菌光解酶三维结构的第一个例子。在这个光解酶分子中发现了两个FAD分子，其中FAD不仅作为催化辅助因子结合到通常的FAD结合位点上，而且还结合到光收集辅助因子的结合位点上。对于来自Anacyctis nidulans的蓝藻光解酶，我们测定了载脂蛋白状态（不含其捕光辅助因子8-HDF）的晶体结构，并研究了催化辅助因子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CRY的功能同源蛋白，我们在2Å分辨率下确定了嗜热蓝藻热共生球菌（Thermosynechococcus elongatus BP-1）的BLUF结构域的晶体结构。在晶体结构的基础上，我们讨论了与关键Tyr8 （FAD-Gln50-Tyr8网络）在结构和功能上连接的Gln50在BLUF蛋白的光诱导光谱位移中的可能作用。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