CELL CYCLE CONTROL AND EXPRESSION OF AP ENDONUCLEASES

细胞周期控制和 AP 核酸内切酶的表达

• 批准号: 6325820
• 负责人: Pablo Arenaz
• 金额: $ 4.04万
• 依托单位: UNIVERSITY OF TEXAS EL PASO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6325820
• 关键词: CHO cells; DNA binding protein; DNA repair; DNA replication; animal genetic material tag; binding sites; cell growth regulation; endonuclease; enzyme activity; gel mobility shift assay; gene expression; genetic disorder; genetic regulation; genetic transcription; glycosidases; molecular cloning; molecular pathology; nucleic acid sequence; polymerase chain reaction; southern blotting; tissue /cell culture; transfection

The long-term goal of this project is to determine the mechanism(s) involved in the temporal regulation of the expression of DNA repair genes. A thorough analysis of the regulatory mechanisms involved will increase our understanding of cell cycle mediated gene regulation in eukaryotes and our knowledge of the DNA repair strategies cell use to ensure self perpetuation without error. The proposed studies have significant implications for examining the relationship between DNA repair and genetic diseases in which predisposition to cancer is a phenotypic characteristic. The temporal modulation of DNA repair processes appears to be a common feature in mammalian cells of different origins. In addition, there appears to be a correlation between altered cell cycle control of DNA repair processes and DNA repair deficiency or hypersensitivity to damage. However, the exact mechanism of this modulation is unclear. It is clear that Chinese Hamster Ovary (CHO) cells are good models for analyzing the regulation of DNA repair processes and extrapolating to the human model. As part of our continuing effort to understand the regulatory mechanism(s) involved in the temporal modulation of DNA repair processes, we propose to examine the control of DNA repair processes at the molecular level. We will use the recently cloned Chinese hamster apurinic/apyrimidinic (AP) endonuclease gene (CHAPE) as a model. We propose to look at the control mechanisms from several different aspects: 1) To examine whether control of DNA repair processes is at the level of transcription. 2) To construct vectors for the characterization of CHAPE gene promoter; 3) To detect the sequence-specific DNA-binding protein in the crude extracts from the hamster cell lines, AA8 and EM9 and also to identify the specific binding sites for these proteins; 4) To stably transfect CHOAA8 and EM9 cells with the APE_I-CAT promoter constructs; and 5) To check the cell cycle inducibility of the CHAPE gene.

该项目的长期目标是确定DNA修复基因表达的时间调节所涉及的机制。对所涉及的调节机制的透彻分析将增加我们对真核生物中细胞周期介导的基因调控的理解，以及我们对DNA修复策略使用细胞使用的了解，以确保自我持续性而不会出错。拟议的研究对检查DNA修复与遗传疾病之间的关系具有重要意义，其中癌症易感性是表型特征。 DNA修复过程的时间调节似乎是不同起源的哺乳动物细胞中的常见特征。另外，DNA修复过程的细胞周期控制与DNA修复缺乏或对损伤的过敏似乎之间似乎存在相关性。但是，此调制的确切机制尚不清楚。显然，中国仓鼠卵巢（CHO）细胞是分析DNA修复过程并推出人类模型的良好模型。 作为我们持续努力了解DNA修复过程涉及的调节机制的一部分，我们建议检查分子水平上DNA修复过程的控制。我们将使用最近克隆的中国仓鼠昆虫/肾上腺素（AP）核酸内切酶基因（CHAPE）作为模型。我们建议从几个不同方面查看控制机制：1）检查DNA修复过程是否处于转录水平。 2）构建矢量以表征Chape基因启动子； 3）检测仓鼠细胞系AA8和EM9的粗提取物中的序列特异性DNA结合蛋白，并确定这些蛋白质的特定结合位点； 4）使用APE_I-CAT启动子构建体稳定转染ChoAA8和EM9细胞； 5）检查Chape基因的细胞周期诱导性。