GENETIC ANALYSIS OF NUCLEOTIDE EXCISION REPAIR

核苷酸切除修复的遗传分析

• 批准号: 6172427
• 负责人: LAWRENCE H THOMPSON
• 金额: $ 43.65万
• 依托单位: UNIVERSITY OF CALIF-LAWRNC LVRMR NAT LAB
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-20 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6172427
• 关键词: CHO cells; DNA binding protein; DNA damage; DNA repair; DNA replication; animal genetic material tag; gene expression; gene mutation; human genetic material tag; molecular genetics; mutant; nucleic acid sequence; nucleotide metabolism; protein purification; protein structure function; radiation genetics; ultraviolet radiation; xeroderma pigmentosum

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.

描述：两个主要目标是：（1）了解突变是如何发生的。 ERCC 2 DNA解旋酶产生两种不同疾病，癌症倾向 着色性干皮病（XP）和发育异常性甲状腺功能异常 (TTD)（2）以ERCC 2为模型，定义其结构-功能关系 解旋酶 ERCC 2是TFIIH转录修复复合物的一个组分， ERCC 2的解旋活性是核苷酸切除所必需的 修复（NER），而不是转录。 在XP组D（XP-D）中， ERCC 2基因通常赋予高紫外线（UV）敏感性，并映射到 含有高度保守结构域的蛋白质区域， 解旋活动。 相反，TTD突变也赋予紫外线敏感性， 但映射到解旋酶结构域外的C末端， 通过影响TFIIH复合物的稳定性来抑制转录缺陷。 使用CHO仓鼠细胞系统，待检验的一般假设是， XP-D突变产生缺乏解旋活性的ERCC 2蛋白，而TTD 突变导致ERCC 2与其他蛋白质的弱关联， TFIIH复合物。 这一概念将通过确定XP是否 突变，而不是TTD突变，可以产生显性表型， 当它们在野生型中过表达时， 仓鼠细胞 为了检验TTD是由弱关联引起的观点， ERCC 2与伴侣蛋白的关系，将确定是否具有UV抗性 可以通过在缺乏正常细胞的情况下过表达TTD突变来恢复。 蛋白 主要假设也将通过纯化正常和 突变ERCC 2从过表达系统，然后进行详细的 纯化蛋白质的表征，以便将功能与 在特定区域发生突变。 这些研究将决定 XP-D蛋白是否以及为什么失去解旋活性，TTD是否 蛋白质保持解旋活性。 一种利用基因内 抑制基因突变将有助于阐明解旋酶之间的关系， 域，并定义与每个域相关联的功能。 这些 机制的见解将指导未来对ERCC 2的结构研究。