MECHANISMS OF MUTAGENIC PROCESSING OF DNA DAMAGE

DNA 损伤的诱变处理机制

• 批准号: 2895905
• 负责人: WILLIAM G MCGREGOR
• 金额: $ 5.25万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 2000-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2895905
• 关键词: DNA damage; DNA repair; DNA replication; HeLa cells; benzopyrenes; drug carcinogenesis; fibroblasts; human genetic material tag; human tissue; neoplasm /cancer genetics; nucleic acid sequence; polymerase chain reaction; proliferating cell nuclear antigen; radiation carcinogenesis; transfection /expression vector; ultraviolet radiation; xeroderma pigmentosum

DESCRIPTION: Xeroderma pigmentosum (XP) patients have an inherited predisposition to sunlight-induced skin cancer, and the cells from the majority of these patients have been shown to be deficient in nucleotide excision repair. Lack of repair of UV-induced DNA damage can explain why these cells are extremely sensitive to mutations induced by UV. However, one class of XP patients, designated variant, are not deficient in excision repair, and yet their cells exhibit an even greater sensitivity to UV-induced mutations than those of classic excision repair-deficient XP patients. Data from UV-induced mutational spectra suggest that the cancer predisposition of XP variant patients results from their cells having a defective DNA replication complex, so that when they replicate DNA containing photoproducts they make frequent errors, and these errors are of a very distinctive kind. Recent data, derived from an in vitro DNA replication assay comparing XP variant and HeLa cell extracts, confirm that this is true. This replication-competent extract from XP variant cells represents the first and, as yet, the only example of a human DNA replication complex that demonstrates error-prone bypass of DNA damage. These cells represent a unique opportunity to investigate the mechanisms by which normal cells achieve fidelity when they replicate damaged DNA, and how errors in replication lead to mutations. Since mutations are causally involved in the process of neoplastic transformation, this study has direct relevance to the study of tumorigenesis. It is proposed to study the mechanism of bypass of a number of lesions by extracts from normal cells and from XP variant cells. It is also proposed to investigate a variety of replication proteins to determine if they may contribute to, or be responsible for, the replication defect in XP variant cells. Specifically, it is proposed: 1) to test whether cell extracts from normal cells give the same mutation spectrum as HeLa cell extracts when replication UV damaged templates in vitro; 2) to expand a current database of UV-induced mutations mediated by replication with XP variant cell extracts; 3) to switch the origin of replication of the replication substrate relative to the target gene, to confirm that XP variant replication complex processes photoproducts asymmetrically, in a strand-specific manner; 4) to test whether the addition of RP-A, RF-C, PCNA, or pol alpha correct the XP variant defect in vitro, and to sequence the coding region of the pol delta gene from 2 XP variant cell lines; 5) to prepare extracts from a variety of XP variant cell lines, to test for complementation; and 6) to examine bypass parameters by normal and XP variant extracts of the following lesions placed site-specifically in the same sequence context: T-T dimers, T-T 6-4s, N2-dG-(+)-trans-anti-benzo[a]pyrene, C8-dG-1-aminopyrine, or C8-dG-2-aminofluorene.

描述：着色性干皮病 (XP) 患者具有遗传性 阳光诱发的皮肤癌的易感性，以及来自 这些患者中的大多数已被证明缺乏核苷酸 切除修复。 紫外线引起的 DNA 损伤缺乏修复可以解释原因 这些细胞对紫外线诱导的突变极其敏感。 然而， 一类 XP 患者，指定为变体，不存在切除缺陷 修复，但它们的细胞表现出更高的敏感性 紫外线诱导的突变比经典切除修复缺陷型 XP 的突变要多 患者。 来自紫外线诱导突变光谱的数据表明，癌症 XP 变异患者的易感性是由于他们的细胞具有 DNA复制复合体有缺陷，因此当它们复制DNA时 包含照片产品的他们经常犯错误，这些错误是 非常有特色的一种。 最新数据，源自体外 DNA 比较 XP 变体和 HeLa 细胞提取物的复制测定，确认 这是真实的。 这种来自 XP 变异细胞的具有复制能力的提取物 代表第一个也是迄今为止唯一的人类 DNA 样本 复制复合物展示了容易出错的 DNA 损伤旁路。 这些细胞提供了一个独特的机会来研究其机制 哪些正常细胞在复制受损 DNA 时能够实现保真度，以及如何实现 复制错误会导致突变。 由于突变是有因果关系的 参与肿瘤转化过程，本研究具有直接意义 与肿瘤发生研究的相关性。 建议研究 通过正常细胞提取物绕过许多病变的机制 来自 XP 变异细胞。 还建议调查各种 复制蛋白以确定它们是否可能有助于或可能是 XP 变异细胞中的复制缺陷是造成这一现象的原因。 具体来说， 建议： 1) 测试正常细胞的细胞提取物是否给出 当复制紫外线受损时，与 HeLa 细胞提取物相同的突变谱 体外模板； 2) 扩展现有的紫外线诱导突变数据库 通过 XP 变异细胞提取物的复制介导； 3）切换 复制底物相对于靶标的复制起点 基因，以确认 XP 变体复制复合物处理光产物 不对称地，以特定链的方式； 4）测试是否添加 RP-A、RF-C、PCNA 或 pol alpha 在体外纠正 XP 变异缺陷， 并对 2 XP 变体的 pol delta 基因的编码区进行测序 细胞系； 5) 制备多种 XP 变异细胞系的提取物， 测试互补性； 6) 正常检查旁路参数 以及以下病变的 XP 变异提取物，特定位置放置在 相同的序列上下文：T-T 二聚体、T-T 6-4s、 N2-dG-(+)-反式-抗苯并[a]芘、C8-dG-1-氨基比林，或 C8-dG-2-氨基芴。