MOLECULAR ANALYSIS OF CARCNINGEN-INDUCED DNA REPAIR

致癌原诱导的 DNA 修复的分子分析

• 批准号: 3175101
• 负责人: STEVEN L DRESLER
• 金额: $ 8.65万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-30 至 1986-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3175101
• 关键词: DNA; DNA repair; acetylation; adenosine triphosphate; chemical carcinogen; chemical carcinogenesis; chromatin; electrophoresis; gel filtration chromatography; human tissue; molecular oncology; nitrosourea; phosphorylation; radiation carcinogenesis; radiotracer; scintillation spectrometry; tissue /cell culture; xeroderma pigmentosum

Excision repair is generally regarded as the major process by which mammalian cells reduce the cytotoxic, mutagenic, and carcinogenic effects of DNA damage produced by ultraviolet radiation (UV) and chemical carcinogens. The object of this proposal is to explore the involvement of ATP and of changes in chromatin structure in excision repair. Previous studies have revealed that, in normal human cells, ATP is required for excision repair of DNA damaged by ultraviolet radiation (UV) at or before the incision step. ATP is also required for repair of UV damage in xeroderma pigmentosum (XP) cells complemented with T4 UV endonuclease. One objective of this proposal is to characaterize specifically the ATP requirement for excision repair in UV endonuclease-complemented XP cells and to compare that requirement enzymologically with the ATP requirement in normal cells. Other ATP-related questions to be studies are: i) whether these ATP requirements are related to involvement of a DNA topisomerase in excision repair; ii) whether any of the incompletely repair deficient XP cell lines show altered ATP requirements; and iii) whether ATP is required for excision repair induced by other carcinogenic DNA damaging agents, e.g., N-methyl-N-nitrosourea (MNU), an alkylating agent, and x-ray, a strand breaking agent. Chromatin changes accompanying repair of UV, MNU and x-ray damage will also be studied: i) determining whether excision repair is accompanied by acetylation, phosphorylation, and/or methylation of specific nuclear proteins; ii) determining at which step in the excision repair pathway poly ADP ribose added to nuclear proteins in response to DNA damage is turned over or degraded; iii) determing whether specific chromatin proteins are gained or lost during the process of excision repair; and iv) determining whether chromatin modifications associated with DNA damage and repair are specifically localized in the regions of chromatin underdoing repair. All of the studies proposed will utilize a permeable cell system in which excision repair proceeds in a fashion essentially identical to that in intact cells, but is accessible at all stages to experimental manipulation. In addition, a novel approach involving biotin-modified dUTP and avidin-agarose chromatography will be used to isolate and directly analyze chromatin undergoing repair. These experiments should generate biochemical data which will be directly applicable to understanding excision repair in vivo, contributing to a more complete molecular description of this important biological process.

切除修复通常被视为主要过程 哺乳动物细胞降低细胞毒性，诱变和致癌作用 紫外线辐射（UV）和化学物质产生的DNA损伤 致癌物。 该提议的目的是探索 ATP和切除修复中染色质结构的变化。 以前的 研究表明，在正常的人类细胞中，需要ATP 切除在因紫外线辐射（UV）或之前损坏的DNA的切除修复 切口步骤。 还需要ATP维修紫外线损伤 甲状腺色素色素（XP）细胞与T4 UV内核酸内切酶相辅相成。 一 该提议的目的是专门为ATP表征 要求在紫外核酸内切酶填充的XP细胞中切除修复 并将酶学上的需求与ATP要求进行比较 正常细胞。 其他与ATP有关的问题是：i）是否 这些ATP要求与DNA拓扑酶的参与有关 切除修复； ii）是否有任何不完全修复缺陷的XP 细胞系显示ATP的改变； iii）是否需要ATP 对于其他致癌DNA损伤剂引起的切除修复， 例如N-甲基-N-硝酸（MNU），烷基化剂和X射线，A 链破裂剂。 紫外线修复的染色质变化 还将研究X射线损坏：i）确定切除是否切除 维修伴随着乙酰化，磷酸化和/或甲基化 特定的核蛋白； ii）确定切除中哪一步 修复途径聚ADP核糖响应于DNA中的核蛋白 损坏已翻转或退化； iii）确定是否具体 在切除过程中获得或丢失染色质蛋白 维修; iv）确定染色质修饰是否与 DNA损伤和维修专门位于 染色质不足修复。 提出的所有研究将利用 可渗透的细胞系统，其中切除以一种方式进行的修复 在完整的单元格中基本相同，但完全可以访问 实验操作的阶段。 另外，一种新颖的方法 涉及生物素改性的DUTP和Avidin-Agarose色谱法将是 用于分离和直接分析染色质进行维修。 这些 实验应生成直接的生化数据 适用于在体内了解切除修复，有助于更多 这一重要生物学过程的完整分子描述。