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控机制(S)。深入分析其中涉及的调控机制将增加我们对真核细胞中细胞周期介导的基因调控的理解，以及我们对DNA修复策略的了解，细胞使用DNA修复策略来确保自我永续而不出错。拟议的研究对于检验DNA修复和遗传性疾病之间的关系具有重要意义，在遗传性疾病中，癌症易感性是一种表型特征。DNA修复过程的时间调制似乎是不同来源的哺乳动物细胞的共同特征。此外，细胞周期改变对DNA修复过程的控制似乎与DNA修复缺陷或对损伤的过敏性有关。然而，这种调节的确切机制尚不清楚。显然，中国仓鼠卵巢(CHO)细胞是分析DNA修复过程调节和推断人类模型的良好模型。作为我们理解DNA修复过程时间调控机制(S)的持续努力的一部分，我们建议在分子水平上研究DNA修复过程的控制。我们将以最近克隆的中国仓鼠无嘌呤/无嘧啶(AP)核酸内切酶基因(CHPE)为模型。我们建议从几个不同的方面来看控制机制：1)检查DNA修复过程是否处于转录水平的控制。2)构建CHAPE基因启动子的表达载体；3)检测仓鼠细胞系aa8和EM9中序列特异的DNA结合蛋白，并确定这些蛋白的特异结合部位；4)将构建的APE_I-CAT启动子稳定地导入CHOAA8和EM9细胞；5)检测CHAPE基因对细胞周期的诱导作用。