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RAD26 FUNCTION IN TRANSCRIPTION COUPLED DNA REPAIR

RAD26 在转录偶联 DNA 修复中的功能

基本信息

批准号：
6386242
负责人：
KEVIN S SWEDER
金额：
$ 15.02万
依托单位：
RUTGERS, THE STATE UNIV OF N.J.
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-08-01 至 2003-07-31
项目状态：
已结题

项目摘要

A fundamental problem faced by all organisms is the maintenance of genomic integrity. DNA damage intergeres with essential cellular processes, and unrepaired or misrepaired DNA can be mutagenic and carcinogenic. Defects in DNA repair associated with human cancer have been found in the cancer-prone diseases xeroderma pigmentation (XP) and nonpolyposis colon cancer. DNA repair proteins encoded by the human genes have yeast homologs which serve analogous functions. Therefore, yeast provides an excellent model system for studying eukaryotic DNA repair and its relationship to the disease state in humans. The overall goal of this proposed study is to elucidate the mechanism of transcription coupled repair in eukaryotes. 1) We will study the effects of altered expression of Rad26 in yeast. Mutations of the RAD26 gene (or ERCC6, its human homolog) results in cells that lack transcription-coupled repair. We will express RAD26 from a regulatable promoter in a rad26 mutant and explore its effect upon transcription-coupled repair and overall repair, under varying expression levels. We will also investigate the expression of a reporter gene Green Fluorescent Protein (GFP), in real time, under the same growth conditions. 2) We will study the effects of transcription rate upon transcription-coupled DNA repair in rad26 mutants. Unlike previous studies with inducible genes, transcription will be continuously increased from very low to very high rates of expression. 3) We will genetically identify factors that interact with Rad26 in vivo using selection for extragenic suppressors and a synthetic lethal screen. The two hybrid system will be used to identify proteins expressed from a yeast genomic library which interact with Rad26 protein. 4) We will investigate the role of chromatin structure in transcription- coupled DNA repair in rad26 mutants and identify domains of Rad26 that direct repair to transcriptionally active chromatin. Our in vivo approach eliminate a major hurdle to present biochemical approaches in that we are investigating the removal of DNA damage from bona fide chromatin substrates.

所有生物体面临的一个根本问题是维护基因组的完整性。DNA损伤与Essential 细胞过程，以及未修复或错误修复的DNA 具有诱变性和致癌性。与DNA修复相关的缺陷已发现与人类癌症有关的易患癌症的疾病干皮病、色素沉着(XP)和非息肉病性结肠癌。人类基因编码的DNA修复蛋白含有酵母菌具有相似功能的同系物。因此，酵母为研究真核dna提供了一个良好的模型系统。修复及其与人类疾病状态的关系。这项拟议研究的总体目标是阐明真核生物转录偶联修复机制。1)我们将研究Rad26在酵母中表达变化的影响。 RAD26基因(或其人类同源基因ERCC6)的突变导致缺乏转录偶联修复的细胞。我们会利用可调控启动子在rad26突变体中表达RAD26 并探讨其对转录偶联修复和整体的影响。修复，在不同的表达水平下。我们还将调查报告基因绿色荧光蛋白的表达 (GFP)，实时，在相同的生长条件下。2)我们会转录速率对转录偶联的影响研究 Rad26突变体的DNA修复。与以前的研究不同的是可诱导基因，转录将不断增加，从从很低到很高的表现率。3)我们将从基因上使用选择识别体内与Rad26相互作用的因子外源抑制物和合成的致命屏障。两个人杂交系统将被用来鉴定表达的蛋白质与RAD26蛋白相互作用的酵母基因组文库。4)我们将研究染色质结构在转录中的作用- Rad26突变体的偶联DNA修复及结构域鉴定 RAD26直接修复转录活性的染色质。我们的体内方法消除了目前生物化学的一个主要障碍我们正在调查去除DNA的方法真正的染色质底物造成的损害。