(6-4) photoproduct photoreactivating enzyme in Drosophila melanogaster

DNA光解酶催化顺式、顺式环丁烷二嘧啶(嘧啶二聚体)的光依赖修复。我们克隆了果蝇光解酶基因的编码基因，它含有一个开放阅读框架，编码61,483个道尔顿蛋白。表达产物在体外具有光修复嘧啶二聚体的活性。经Southern杂交分析，克隆的基因与多线染色体44C-D带、光修复缺陷(phr-)基因的遗传位置以及phr-基因的DNA杂交，后者表明phr-突变基因中存在DNA重排(S)。这些结果表明，所克隆的基因与果蝇的phr基因相对应。我们发现果蝇的光解酶活性在胚胎和成体卵巢中都很高，约为6700个分子/细胞，而光解酶的mRNA只在成体卵巢中丰富，这表明果蝇光解酶基因是母体基因。我们还从果蝇胚胎中纯化了光解酶。经十二烷基硫酸钠-凝胶电泳法测得其相对分子质量为62,000。纯化的光解酶对紫外光照射下的质粒DNA的光复活的作用光谱在440 nm附近有一个单一的尖峰。预测的果蝇光解酶的氨基酸序列与金鱼光解酶的氨基酸序列有广泛的同源性，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.