MOLECULAR CLONING OF A RADIATION REPAIR GENE XRCC5

真核生物对DNA损伤剂的反应，如电离 辐射，是由多种DNA修复的有效性决定的 系统。有缺陷的修理，以能力减弱的形式 准确维修或通过不准确的维修过程，可能会导致 涉及负责细胞控制的基因的突变 扩散。了解这些修复过程的本质是 对于阐明随后通过哪些机制 对健康的不利影响，如突变、胎儿畸形和癌症 都被表达出来。电离辐射诱发多种类型的DNA损伤 哺乳动物细胞包括DNA单链断裂(SSB)、DNA双断裂 链断裂(DSB)、DNA-蛋白质交联链、DNA-DNA链间 交联物和改变的DNA碱基。尽管-很明显，人类 细胞可以修复这些损伤中的许多，但几乎没有详细的知识 基因的性质和控制这些基因的编码酶 维修流程。为了了解DNA修复是如何调节的 辐射敏感性、癌基因表达与重组相关 遗传性疾病，辐射特异的DNA修复基因必须分离 并以此为特征。 在本项目的当前资金期内，一个全面的 战略和人类定位克隆的所有基本基础 本实验室已建立了DSB修复基因XRCC5。因此， 此续期申请的主要目标是继续努力 人XRCC5基因的分离与鉴定。具体的 目的是：1)人类XRCC5基因在2号染色体上的区域定位(在 百万级)；2)修复熟练的X射线杂交种的构建 面板，每个杂交包含人类2号染色体的一个片段(1-3Mb) 已分配XRCC5的位置；3)候选XRCC5的位置克隆 重组克隆及其功能分析；4)分子 克隆的XRCC5基因的鉴定。我们相信这一步- 利用先进的分子遗传技术的STEP方法将 极大地促进了第一次人类辐射修复的隔离 负责电离辐射诱导DNA重新连接的基因(S) 双链断裂。

The response of eukaryotes to DNA damaging agents, such as ionizing radiation, is determined by the effectiveness of a variety of DNA repair systems. Defective repair, in the form of a diminished capacity for accurate repair or through inaccurate repair processes, can lead to mutations involving genes responsible for the control of cell proliferation. Understanding the nature of these repair processes is central to the elucidation of the mechanisms through which subsequent adverse health effects such as mutations, fetal malformations, and cancers are expressed. Ionizing radiation induces various types of DNA damage in mammalian cells including DNA single-strand breaks (SSB), DNA double strand breaks (DSB), DNA-protein crosslinks, DNA-DNA interstrand crosslinks, and altered DNA bases. Although- it is obvious that human cells can repair many of these lesions, there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. ln order to understand how DNA repair modulates radiation sensitivity, oncogene expression, and recombination-related genetic diseases, radiation-specific DNA repair genes have to be isolated and characterized. During the current funding period of this project, a comprehensive strategy and all the basic grounds for the positional cloning of a human DSB repair gene XRCC5 have been established in our laboratory. Thus, the primary objective of this renewal application is to continue our efforts on the isolation and characterization of human XRCC5 gene. The specific aims are: 1) Regional localization of human XRCC5 gene in chromosome 2 (at the megabases level); 2) Construction of a repair-proficient X-ray hybrid panel, each hybrid containing a fragment of human chromosome 2 (1-3 Mb) where the XRCC5 has been assigned ;3) Position cloning of candidate XRCC5 recombinant clones and functional analysis of these clones; 4) Molecular characterization of the cloned XRCC5 gene. We believe that this step-by- step approach utilizing advanced molecular genetic technologies will substantially facilitate the isolation of the first human radiation repair gene(s) responsible for the rejoining of ionizing radiation-induced DNA double-strand breaks.