ENZYMOLOGY AND GENETICS OF DROSOPHILA DNA REPAIR

该计划的最终目标是构建一个体外系统。 由确定的蛋白质组成，用于研究高等生物 真核生物与DNA损伤作斗争。果蝇将受雇于 这些研究，因为一个平行的基因分析将被执行到 还定义了正在研究的酶的体内功能。自.以来 可用的突变体和我们希望分离的突变体是人类的类似物 修复相关障碍，这些研究将提供相关证据 人类DNA修复及其在突变、癌症发生和发展中的作用 重组。 DNA代谢中几种关键酶的分离与鉴定 将会被承担。这些酶包括DNA聚合酶Beta，DNA 聚合酶-γ，两种依赖于ATP的脱氧核糖核酸酶，一种类似Recc A的酶 蛋白质和损伤特异的核酸内切酶。根据它们的酶学 特征，这些酶的缺陷将在 现有的修复缺陷突变体。选定的基因也将被克隆 通过筛选cDNA表达文库或通过筛选基因组文库 用混合序列寡核苷酸探针。克隆人将被用于 研究这些基因的正常调节，并将这种调节改变为 确定它们在DNA修复、重组和合成中的作用。克隆人 也将被用来指导一项基因研究，这将进一步确定 蛋白质在体内的功能。恢复的克隆将提供 有可能用来恢复相似基因的探针 人类基因组。还将研究线粒体DNA新陈代谢。 努力开发一种体外模型系统。

The ultimate goal of this program is to construct an in vitro system composed of defined proteins to study the mechanisms employed by higher eukaryotes to contend with DNA damage. Drosophila will be employed in these studies, because a parallel genetic analysis will be performed to also define the in vivo function of the enzymes under investigation. Since the available mutants and those we expect to isolate are analogues of human repair-related disorders, these studies will provide evidence relevant to human DNA repair and its involvement in mutagenesis, carcinogenesis, and recombination. The isolation and characterization of several key enzymes of DNA metabolism will be undertaken. These enzymes include DNA polymerase Beta, DNA polymerase Gamma, two ATP-dependent deoxyribonucleases, a rec A-like protein, and a damage-specific endonuclease. Following their enzymological characterization, defects in these enzymes will be sought among the available repair-deficient mutants. Selected genes will be cloned either by screening a cDNA expression library or by screening genomic libraries with mixed-sequence oligonucleotide probes. The clones will be employed to study the normal regulation of these genes and to alter that regulation to define their role in DNA repair, recombination and synthesis. The clones will also be employed to guide a genetic study which will further define the in vivo function of the proteins. The recovered clones will provide probes which can potentially be employed to recover analogous genes from the human genome. Mitochondrial DNA metabolism will also be investigated in an effort to develop a model in vitro system.