DISTRIBUTION OF UV DAMAGE AND REPAIR IN MAMMALIAN CELLS

哺乳动物细胞中紫外线损伤和修复的分布

• 批准号: 3254247
• 负责人: DAVID L MITCHELL
• 金额: $ 16.43万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 1995-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3254247
• 关键词: DNA damage; DNA repair; chromatin; gel mobility shift assay; genome; human tissue; laboratory rat; photobiology; radiation carcinogenesis; southern blotting; ultraviolet radiation

Sunlight-induced skin cancer is a major public health concern. Since the discovery of structural damage in UV-irradiated DNA 25 years ago, the molecular mechanisms underlying photocarcinogenesis have been a major focus of investigation. And yet, the molecular events that mitigate the effects of UV damage in DNA, and determine their pathogenic consequences in higher eukaryotes, are not well understood. Immunological and biochemical techniques have revealed a diverse spectrum of photodamage in DNA; each lesion may have its own cytotoxic and mutagenic potential and may be modulated by distinct DNA repair mechanisms. Recently, new approaches have been developed to measure DNA repair in specific sequences and have been used to describe the preferential excision of cyclobutane dimers in transcribing genes. As data accumulate, it is evident that many factors affect DNA repair in specific genes and that other photoproducts, in addition to the cyclobutane dimer, may be biologically important. In this regard, the (6-4) photoproduct has gained much attention over the past few years as a significant lethal and mutagenic determinant in UV-irradiated DNA. However, little is known regarding the induction and repair of this lesion in specific regions of mammalian chromatin. It is our goal to develop new approaches and exploit existing techniques to quantify (6-4) photoproducts in human and rodent cells. We will adapt the technique of Bohr and Hanawalt to the analysis of (6-4) photoproducts in DNA as "photoinduced alkali-labile sites" (PALS) using Southern transfer and hybridization to specific probes. In addition, we will modify this procedure to quantify (6-4) photoproducts in specific sequences as antibody-binding sites using a mobility shift immunoassay. We will use these techniques to evaluate preferential (6-4) photoproduct repair in mammalian genes and investigate how DNA structure and function affect its formation and excision. In addition, we will use radioimmunoassays to compare the kinetics of (6-4) photoproduct repair in functionally-defined regions of DNA (i.e., nuclear matrix and DNase I-sensitive domains of chromatin). Along with established techniques for analyzing cyclobutane dimers in specific DNA sequences, these novel procedures will be used to investigate the function of the human ERCC2 gene and determine its role in mammalian DNA excision repair. By characterizing the fine structure of (6-4) photoproduct induction and repair in mammalian cells, we hope to elucidate the lethal and mutagenic mechanisms of UV light and further our understanding of the molecular basis of carcinogenesis.

阳光引起的皮肤癌是一个主要的公共卫生问题。 以来 25年前发现紫外线照射DNA的结构损伤， 光致癌作用的分子机制一直是 调查的重点。 然而，那些减轻了 紫外线对DNA损伤的影响，并确定其致病后果 在高等真核生物中，还没有很好的理解。 免疫学和 生物化学技术揭示了光损伤的多样性 在DNA中;每个损伤可能具有其自身的细胞毒性和致突变潜力 并且可以通过不同的DNA修复机制来调节。 近日，新 已经开发了一些方法来测量特定细胞中的DNA修复， 序列，并已被用来描述优先切除的 环丁烷二聚体在转录基因中的作用。 随着数据的积累， 显然，许多因素影响特定基因中的DNA修复， 除了环丁烷二聚体之外， 生物重要性。 在这方面，（6-4）光产物具有 在过去的几年里，作为一种重要的致命武器， 紫外线照射的DNA中的诱变决定簇。 然而， 关于这种病变在特定区域的诱导和修复， 哺乳动物的染色质 我们的目标是开发新的方法， 利用现有技术量化人体内的（6-4）光产物， 啮齿动物细胞 我们将使玻尔和哈纳沃特的技术适用于 DNA中（6-4）光产物作为“光诱导碱不稳定”的分析 利用Southern转移和杂交技术， probes. 此外，我们将修改此程序以量化（6-4） 在特定序列中的光产物作为抗体结合位点， 迁移率变动免疫测定法 我们将使用这些技术来评估 优先（6-4）光产物修复在哺乳动物基因和调查 DNA结构和功能如何影响其形成和切除。 在 此外，我们将使用放射免疫测定来比较（6-4）的动力学。 在DNA的功能限定区域中的光产物修复（即，核 基质和染色质的DNA酶I敏感结构域）。 沿着 已建立的分析特定DNA中环丁烷二聚体的技术 序列，这些新的程序将被用来调查 人ERCC 2基因的功能，并确定其在哺乳动物DNA中的作用 切除修复 通过表征（6-4）的精细结构， 在哺乳动物细胞中的光产物诱导和修复，我们希望 阐明紫外线的致死和致突变机制，并进一步研究紫外线对小鼠的致死和致突变作用。 了解致癌的分子基础。