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: 2683460
• 负责人: PHILIP COURTLAND HANAWALT
• 金额: $ 88.11万
• 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/2683460
• 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.

这个项目涉及癌症的分子流行病学， 首先是意识到多达六个独立的 遗传和表观遗传事件可能参与了从 从正常细胞转变为恶性细胞。基因组DNA的变化发生在特定的 并可导致原癌基因的激活或失活 肿瘤抑制基因通过突变、重组、基因 染色体扩增、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的增加 与受损DNA的修复和/或复制中的缺陷相关。 我们的最终目标是了解受损DNA的处理过程 在哺乳动物细胞中与癌症的发生有关。 在开发敏感技术方面具有开拓性 定量特定DNA损伤的限制性内切酶片段 基因组的区域我们将扩展我们的基因组内精细分析 DNA修复的结构，了解控制效率的因素 在染色质中的过程以及在不同的功能域中 基因组，如复制起源和转录的表达基因 不同的RNA聚合酶。发现了优先修复的 转录的DNA链，我们将测试一个模型 基于增强转录因子的转录偶联修复 通过RNA聚合酶II的5‘-3’核酸外切酶活性缩短。 将关键地测试链特异性DNA修复可以 作为一种敏感的转录检测方法。与核基质相关的 将对DNA进行表征，以确定这是否是 转录偶联修复。干皮病的有限区域修复 色素化，补充性C组，将评估学习的基础 对于癌症易感表型，以及修复缺陷的表达 将对Cockayne综合征的基因进行研究，以了解 有缺陷且没有癌症倾向。修复中的差异 处于危险中的特定基因可能解释了一些深刻的 不同组织致癌反应的差异 相同的组织在不同的生物体中。携带病变的质粒探针 已定义的位点将用于分析特定的序列修复 不同遗传背景的细胞。已定义的嵌合质粒将 也可用于将基因导入不同的基因组结构域进行研究 损害处理的特定特征导致增强 损伤的DNA在人类细胞中的整合。我们还将探索 非增殖性细胞DNA周转在非增殖性脑损伤中的作用 突变的精细结构来验证我们的假设转录- 相关的DNA周转可能导致异常高的突变频率 在某些领域。 这项研究将大大有助于我们对 某些癌症易感人群DNA损伤处理缺陷的基础 遗传性疾病，也应该导致新的，敏感的探查 对人体细胞损伤和修复的分析。另外，我们的研究 应该有助于解释DNA损伤和DNA周转在 生物终点，如生存、突变和致癌。