(6-4) photoproduct photoreactivating enzyme in Drosophila melanogaster

DNA光解酶催化顺式、同环丁烷二嘧啶（嘧啶二聚体）的光依赖性修复。我们克隆了果蝇光解酶基因cDNA；它包含一个开放的阅读框来编码一个61483道尔顿蛋白。大肠杆菌细胞合成的cDNA产物在体外表现出光修复嘧啶二聚体的活性。克隆的cDNA经southern blot分析与多烯染色体44C-D带、光修复缺陷基因（phr-）的遗传定位位点和phr-基因的DNA杂交；后者揭示了phrr突变基因内部存在DNA重排。这些结果表明，克隆的cDNA与果蝇的phr基因相对应。我们发现果蝇的光解酶在胚胎和成年卵巢中都有很高的活性（约6700分子/细胞），而光解酶mRNA仅在成年卵巢中丰富，这表明果蝇的光解酶基因是一个母源基因。我们还从果蝇胚胎中纯化了光解酶。sds -凝胶电泳估计分子量为62000。纯化的光解酶用于紫外辐照质粒DNA的光再激活，其作用谱在440 nm左右显示出一个单一的锐最大值。预测的果蝇光解酶氨基酸序列与金鱼鲫鱼光解酶具有广泛的同源性，但与微生物的同源性有限。

DNA photolyase catalyzes the light-dependent repair of cis, syn-cyclobutane dipyrimidines (pyrimidine dimers). We cloned cDNA encoding the Drosophila photolyase gene ; it contained an open reading frame to encode a 61,483 Dalton protein. The product of the cDNA made in E.coli cells showed activity to photorepair pyrimidine dimers in vitro. The cloned cDNA hybridized to the band 44C-D of the polytene chromosome, genetically mapped site of photorepair-deficient (phr-) gene, and also to DNA of the phr-gene by the southern blot analysis ; the latter revealed that the phr-mutant gene has DNA rearrangement (s) within it. These results indicate that the cloned cDNA corresponds to the phr gene of Drosophila. We found that activity of photolyase of Drosophila is very high in the embryo-about 6700 molecules/cell-and also in the adult ovary whereas mRNA of photolyase is abundent only in the adult ovary, an indication that the Drosophila photolyase gene is a maternal gene. We have also purified Drosophila photolyase from embryo. A molecular weight of 62,000 was estimated from SDS-gel electrophoresis. The action spectrum of the purified photolyase for photoreactivation of UV-irradiated plasmid DNA showed a single, sharp maximum around 440 nm. The predicted amino-acid sequence of Drosophila photolyase shares an extensive homology with that of a goldfish Carassius auratus photolyase but only limited homologies with those of microorganisms.