ENZYMOLOGY AND GENETICS OF DROSOPHILA DNA REPAIR

本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，以研究高等生物学的机制。 真核生物来对抗DNA损伤。 果蝇将被用于 这些研究，因为将进行平行的遗传分析， 还定义了所研究的酶的体内功能。 以来 可用突变体和我们期望分离的突变体是人类的类似物， 修复相关疾病，这些研究将提供相关证据， 人类DNA修复及其在诱变、致癌和 重组 几种DNA代谢关键酶的分离及性质研究 将进行。 这些酶包括DNA聚合酶β，DNA 聚合酶γ，两种ATP依赖性脱氧核糖核酸酶，一种recA样 蛋白和损伤特异性内切核酸酶。 根据他们的酶学 表征，这些酶中的缺陷将在 修复缺陷突变体。 选定的基因将被克隆， 通过筛选cDNA表达文库或通过筛选基因组文库 混合序列寡核苷酸探针。 这些克隆人将被用来 研究这些基因的正常调节，并改变这种调节， 定义它们在DNA修复、重组和合成中的作用。 克隆人 还将用于指导遗传研究， 蛋白质的体内功能。 恢复的克隆体将提供 可以潜在地用于从DNA中回收类似基因的探针， 人类基因组 线粒体DNA代谢也将进行研究 以努力开发体外系统模型。

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.