DOMAIN ORGANIZATION OF DNA REPAIR IN HUMAN CELLS

通过核苷酸切除修复（NER），生物体去除受损的 核苷酸，例如UV诱导的环丁烷嘧啶二聚体。 NER有两个子途径：全局基因组修复（GGR）和转录 偶联修复（TCR）。有证据表明TCR更有效， 发生得早。显然，RNA聚合酶在受损的DNA和 转录阻断损伤从转录链的表达 基因被移除。虽然对GGR和TCR的遗传控制了解很多， 关于它们的细胞定位知之甚少。调查人员将 使用5-碘脱氧尿苷（IdU）标记人成纤维细胞中的修复斑 在GGR（着色性干皮病-C）或TCR（Cockayne's 综合征），以确定DNA合成的焦点位点。脉冲标记 将进行实验以识别和计数早期和晚期病灶， 修复合成使用不同的胸苷类似物， 绘制修复的早期和晚期结构域，并将其与染色体 基因密度高和低的区域。他们还将分析相对 在修复灶的间期核中的分布和共定位， GGR和TCR的诊断蛋白。预计这些细胞学 方法将补充NER的基本生物化学理解， 为人类细胞中GGR和TCR的调节提供了新的见解。

Through nucleotide excision repair (NER), an organism removes the damaged nucleotides e.g. UV-induced cyclobutane pyrimidine dimers from its genome. There are two sub-pathways of NER; global genome repair (GGR) and transcription coupled repair (TCR). There is some evidence that TCR is more efficient and occurs early. Apparently, RNA polymerase stops at the damaged DNA and the transcription blocking lesion from the transcribed strands of the expressed gene is removed. While much is known about the genetic control of GGR and TCR, very little is known about their cellular localization. The investigators will use 5-iododeoxyuridine (IdU) labeling of repair patches in human fibroblasts with deficiencies either in GGR (Xeroderma Pigmentosa-C) or in TCR (Cockayne's syndrome) to determine the focal sites of DNA synthesis. Pulse-labeling experiments will be done to identify and enumerate the early and late foci of repair synthesis. Using different thymidine analogs, the investigators expect to map the early and late domains of repair and to relate these to chromosomal regions of high and low gene density. They will also analyze the relative distribution and co-localization in interphase nuclei of repair foci and proteins diagnostic of GGR and TCR. It is anticipated that these cytological approaches will complement the basic biochemical understanding of NER and provide new insights into the regulation of GGR and TCR in human cells.