CELLULAR PROCESSING OF DAMAGED DNA--ROLE IN ONCOGENESIS

受损 DNA 的细胞处理——在肿瘤发生中的作用

• 批准号: 2390687
• 负责人: PHILIP COURTLAND HANAWALT
• 金额: $ 101.86万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-06-05 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2390687
• 关键词: CHO cells; DNA damage; DNA directed RNA polymerase; DNA repair; Escherichia coli; carcinogenesis; electroporation; genetic transcription; nuclear matrix; plasmids; xeroderma pigmentosum

This project is concerned with the molecular epidemiology of cancer, beginning with the realization that as many as half a dozen independent genetic and epigenetic events may be involved in the transformation from a normal cell to malignancy. The changes in genomic DNA occur at specific sites and can lead to activation of protooncogenes or inactivation of tumor suppressor genes through mutation, recombination, gene amplification, translocation, or other chromosomal abnormalities. In some human hereditary diseases an increased incidence of neoplasia is correlated with a defect in the repair and/or replication of damaged DNA. Our ultimate objective is to understand how the processing of damaged DNA in mammalian cells relates to carcinogenesis. Having pioneered in the development of sensitive techniques for quantifying particular DNA lesions in restriction fragments from specific regions of the genome we will extend our analysis of intragenomic fine structure of DNA repair, to learn the factors that control the efficiency of the process in chromatin and in different functional domains of the genome, such as replication origins and expressed genes transcribed by different RNA polymerases. Having discovered preferential repair of the transcribed DNA strand in expressed genes, we will test a model for transcription-coupled repair based upon factors that enhance transcript shortening by the 5'- 3' exonuclease activity of RNA polymerase II. We will critically test the possibility that strand-specific DNA repair can be used as a sensitive assay for transcription. Nuclear matrix associated DNA will be characterized to determine whether that is the site of transcription-coupled repair. Domain limited repair in xeroderma pigmentosum, complementation group C, will be assessed to learn the basis for the cancer prone phenotype, and the deficiency in repair of expressed genes in Cockayne's syndrome will be studied to understand the basis for the defect and the absence of cancer proneness. Differences in the repair of particular genes at risk may account for some of the profound differences seen in the carcinogenic responses of different tissues and of the same tissue in different organisms. Plasmid probes carrying lesions at defined sites will be used in the analysis of specific sequence repair in cells of different genetic background. The defined chimeric plasmids will also be used to introduce genes into different genomic domains to study the specific features of damage processing that result in the enhanced integration of damaged DNA in human cells. We will also explore the possible role of localized DNA turnover in non-proliferating cells in the fine structure of mutagenesis to test our hypothesis that transcription- associated DNA turnover may result in anomalous high mutation frequencies in some domains. This research should contribute substantially to our understanding of the basis for DNA damage processing deficiencies in certain cancer-prone hereditary diseases and it should also result in new, sensitive probes for the analysis of damage and repair in human cells. In addition, our studies should help to interpret the role of DNA damage and DNA turnover in biological end points such as survival, mutagenesis, and carcinogenesis.

该项目涉及癌症的分子流行病学， 首先是认识到多达六个独立的 遗传和表观遗传事件可能参与从 正常细胞转变为恶性肿瘤。基因组DNA的变化发生在特定的时间 位点，并可导致原癌基因的激活或失活 抑癌基因通过突变、重组、基因 扩增、易位或其他染色体异常。在一些 人类遗传性疾病肿瘤发病率增加 与受损 DNA 的修复和/或复制缺陷相关。 我们的最终目标是了解受损 DNA 的加工过程 在哺乳动物细胞中与致癌作用有关。 率先开发敏感技术 定量特定限制性片段中的特定 DNA 损伤 我们将扩展基因组内精细分析的基因组区域 DNA修复结构，了解控制效率的因素 染色质和不同功能域中的过程 基因组，例如复制起点和转录的表达基因 不同的RNA聚合酶。发现优先修复 在表达基因中转录 DNA 链，我们将测试一个模型 基于增强转录因子的转录耦合修复 RNA 聚合酶 II 的 5'- 3' 核酸外切酶活性导致的缩短。我们 将严格测试链特异性 DNA 修复的可能性 用作转录的灵敏测定。核基质相关 DNA 将被表征以确定这是否是 转录耦合修复。干皮病的域有限修复 色素，补充组C，将被评估以学习基础 对于癌症倾向表型，以及表达的修复缺陷 将研究科凯恩综合症的基因，以了解其基础 缺陷和不存在癌症倾向。修复上的差异 处于危险中的特定基因可能解释了一些深远的影响 不同组织和细胞的致癌反应存在差异 不同生物体内的相同组织。携带损伤的质粒探针 定义的位点将用于特定序列修复的分析 不同遗传背景的细胞。定义的嵌合质粒将 也可用于将基因引入不同的基因组结构域以进行研究 导致增强的损伤处理的具体特征 受损DNA在人体细胞中的整合。我们还将探索 局部 DNA 周转在非增殖细胞中的可能作用 诱变的精细结构来检验我们的假设，即转录- 相关的 DNA 周转可能导致异常的高突变频率 在某些领域。 这项研究应该会极大地促进我们对 某些易患癌症的 DNA 损伤处理缺陷的基础 遗传性疾病，它还应该产生新的、灵敏的探针 分析人体细胞的损伤和修复。此外，我们的研究 应该有助于解释 DNA 损伤和 DNA 更新的作用 生物学终点，例如生存、诱变和致癌。