GENETIC ANALYSIS OF NUCLEOTIDE EXCISION REPAIR

描述：两个主要目标是：(1)了解基因突变是如何 ERCC2 DNA解旋酶产生两种不同疾病，易患癌症 色素性干皮病与发育性毛发硫代营养不良 (TTD)和(2)将ERCC2的结构-功能关系定义为模型 解旋酶。ERCC2是TFIIH转录修复复合体的一个组成部分， 而ERCC2的解离活性是核苷酸切除所必需的 修复(NER)，但不转录。在XP组D(XP-D)中， ERCC2基因通常具有较高的紫外线敏感度，并映射到 含有高度保守结构域的蛋白质区域，负责 解除活动。相反，TTD突变也使人对紫外线敏感。 但映射到解旋酶结构域之外的C-末端，可能会导致微妙的 转录缺陷通过影响TFIIH复合体的稳定性。 使用CHO仓鼠-细胞系统，要检验的一般假设是 XP-D突变产生缺乏解卷活性的ERCC2蛋白，而TTD 突变导致ERCC2与细胞中其他蛋白的弱结合 TFIIH情结。这一概念将通过确定XP 突变，但不是TTD突变，可以产生显性表型，这 当它们在野生型中过度表达时，表现为紫外线敏感性 仓鼠细胞。要检验TTD是弱关联的结果这一观点 ERCC2与配对蛋白的结合，将确定是否具有紫外线抗性 在缺乏正常的情况下，可以通过过度表达TTD突变来恢复 蛋白。主要假设也将由Purifin Normal和Purifin Normal进行验证 突变的ERCC2，然后进行详细的 用于功能关联的纯化蛋白的鉴定 随着特定区域的突变而变化。这些研究将确定 XP-D蛋白是否以及为什么失去解离活性，以及TTD是否 蛋白质保持解离活性。一种利用基因内标记的新方法 抑制子突变有助于阐明解旋酶之间的关系 并定义与每个域相关联的函数(S)。这些 机械论的见解将指导未来对ERCC2的结构研究。

DESCRIPTION: The two main goals are to: (1) Understand how mutations in the ERCC2 DNA helicase produce two different diseases, cancer-prone xeroderma pigmentosum (XP) and developmentally abnormal trichothiodystophy (TTD), and (2 Define structure-function relationships of ERCC2 as a model helicase. ERCC2 is a component of the TFIIH transcription-repair complex, and the unwinding activity of ERCC2 is required for nucleotide excision repair (NER) but not transcription. In XP group D (XP-D), mutations in the ERCC2 gene generally confer high ultraviolet (UV) sensitivity and map to regions of the protein containing highly conserved domains responsible for unwinding activity. In contrast, TTD mutations also confer UV sensitivity but map to the C-terminus outside the helicase domains and may cause subtle transcription defects by affecting the stability of the TFIIH complex. Using a CHO hamster-cell system, the general hypothesis to be tested is that XP-D mutations produce ERCC2 proteins that lack unwinding activity while TTD mutations result in weak association of ERCC2 with other proteins in the TFIIH complex. This concept will be tested by determining whether XP mutations, but not TTD mutations, can produce a dominant phenotype, which manifests as UV sensitivity, when they are overexpressed in wild-type hamster cells. To examine the idea that TTD results from a weak association of ERCC2 with partner proteins, it will be determined whether UV resistance can be restored by overexpressing TTD mutations in the absence of normal protein. The main hypothesis will also be tested by purifyin normal and mutant ERCC2 from an overexpression system, and then performing detailed characterization of purified proteins in order to correlate functiona changes with mutations in specific domains. These studies will determine whether and why XP-D proteins lose unwinding activity and whether TTD proteins retain unwinding activity. A novel approach using intragenic suppressor mutations will help to elucidate relationships among the helicase domains and to define the function(s) associated with each domain. These mechanistic insights will guide future structural studies on ERCC2.