RAD26 FUNCTION IN TRANSCRIPTION COUPLED DNA REPAIR

RAD26 在转录偶联 DNA 修复中的功能

• 批准号: 6181053
• 负责人: KEVIN S SWEDER
• 金额: $ 14.58万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6181053
• 关键词: DNA binding protein; DNA damage; DNA repair; binding proteins; chimeric proteins; chromatin; fungal genetics; gene expression; genetic library; genetic models; genetic promoter element; genetic transcription; green fluorescent proteins; intermolecular interaction; lethal genes; mutant; reporter genes; suppressor mutations; yeast two hybrid system; yeasts

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.

所有生物面临的一个基本问题是 基因组的完整性。 DNA损伤与重要的 细胞过程，未修复或错误修复的DNA可能会 致突变和致癌。 DNA修复相关缺陷 与人类癌症有关联的癌症易发性疾病 干皮病色素沉着（XP）和非息肉病性结肠癌。 由人类基因编码的DNA修复蛋白具有酵母 具有类似功能的同系物。 因此，酵母 为研究真核DNA提供了一个极好的模型系统 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 真核生物中的转录偶联修复机制。 1)我们 将研究酵母中Rad 26表达改变的影响。 RAD 26基因（或ERCC 6，其人类同源物）的突变 导致细胞缺乏转录偶联修复。 我们将 在rad 26突变体中从可调控启动子表达RAD 26 并探讨其对转录偶联修复和整体 修复，在不同的表达水平。 我们亦会研究 报告基因绿色荧光蛋白的表达 (GFP)，在真实的时间内，在相同的生长条件下。 2)我们将 研究转录速率对转录偶联的影响， rad 26突变体中的DNA修复。 与以往的研究不同， 诱导基因，转录将不断增加， 非常低到非常高的表达率。3)我们将从基因上 使用选择法鉴定与Rad 26在体内相互作用因子， 基因外抑制子和合成致死筛选。 两 杂交系统将被用于鉴定从一种 与Rad 26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- 在rad 26突变体中偶联DNA修复，并鉴定 Rad 26，其直接修复转录活性染色质。 我们 体内方法消除了目前生物化学 我们正在研究如何去除DNA 真正的染色质底物造成的损伤。