Oxidative Damage to DNA Repair Pathways

DNA 修复途径的氧化损伤

• 批准号: 6747655
• 负责人: CAMERON J KOCH
• 金额: $ 30.43万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-12 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6747655
• 关键词: CHO cells; DNA repair; binding sites; biochemistry; cytotoxicity; enzyme mechanism; glucose 6 phosphate dehydrogenase deficiency; immunoprecipitation; oxidation reduction reaction; oxidative stress; radiation sensitivity; sulfides; thiols; western blottings

DESCRIPTION (provided by applicant): Redox regulation is a term describing modification of function by a change in the state of oxidation vs. reduction of critical control molecules, usually proteins. When cells are subjected to reactive oxygen species in a process described as 'oxidative stress', pathways susceptible to redox regulation may be altered. Defining the mechanism of such alteration is often difficult because oxidative stress can additionally cause damage to multiple cellular targets, including membrane, organelles and chromatin, often leading to mitotic or apoptotic death. Our proposed experiments will allow a separation of damage to the redox-regulated proteins from that of damage to targets of cytotoxicity. Our hypothesis is that alterations in the redox status of DNA repair proteins may impair DNA damage repair. In order to cause the mild and specific oxidation of protein thiols we employ the disulfide of mercaptoethanol, hydroxy-ethyldisulfide (HEDS). Normal cells are able to prevent thiol-disulfide exchange of their protein and non-protein thiols with HEDS via reducing equivalents produced by the pentose cycle, whose key regulatory enzyme is glucose-6-phosphate-dehydrogenase (G6PD). To prevent this, we investigated a CHO cell line without G6PD activity (E89). Reversibility of observed effects was established by transfecting the G6PD gene back into the E89 mutant. With this model system, we will demonstrate that radiation sensitization, inhibition of DNA repair and inhibition of Ku binding to DNA ends are all caused by incubation of E89 cells with non-toxic concentrations of HEDS. These effects are not seen in parental cells or A1A transfectants. Just as 'p53' may be the 'guardian of the genome' we suggest that G6PD is the 'protector of proteins'. This Application will test this interesting concept using combined biochemical and genetic approaches. Specific Aim 1 will determine the kinetic relationships between HEDS mediated radiosensitization and biochemical modulation. Multiple genetic and biochemical tests will determine the specific sensitivity of Ku to oxidative modification by HEDS treatment. Specific Aim 2 will determine the (bio)chemical mechanism of HEDS oxidation of cellular protein thiols. Specific Aim 3 will investigate other aspects of DNA repair and DNA structural organization to determine their sensitivity to redox regulation.

描述（由申请人提供）：氧化还原调节是一个术语，描述了通过氧化状态与关键对照分子（通常是蛋白质）的减少状态的变化来修饰功能的术语。当细胞在称为“氧化应激”的过程中受到活性氧，易受氧化还原调节的途径。定义这种改变的机制通常很困难，因为氧化应激可能会对包括膜，细胞器和染色质在内的多个细胞靶标造成损害，这通常会导致有丝分裂或凋亡死亡。我们提出的实验将允许将氧化还原调节的蛋白与细胞毒性靶标的损害分开。我们的假设是，DNA修复蛋白的氧化还原状态的改变可能会损害DNA损伤修复。为了引起蛋白质硫醇的轻度和特异性氧化，我们采用了墨塔乙醇，羟基乙醇（HEDS）的二硫化物。正常细胞能够通过还原五棵循环产生的等效物来防止硫醇二硫化物与其蛋白质和非蛋白硫醇与HEDS的交换，五旬节循环的关键调节酶是葡萄糖-6-磷酸葡萄糖 - 脱水酶（G6PD）。为了防止这种情况，我们研究了没有G6PD活性的CHO细胞系（E89）。通过将G6PD基因转染回E89突变体来确定观察到的效果的可逆性。使用此模型系统，我们将证明辐射敏化，抑制DNA修复和KU结合与DNA末端的抑制都是由E89细胞孵育的HEDS无毒浓度引起的。这些作用在亲本细胞或A1a转染物中没有看到。就像“ p53”可能是“基因组的监护人”一样，我们也建议G6PD是“蛋白质的保护者”。该应用将使用合并的生化和遗传方法来测试这个有趣的概念。具体目标1将确定HEDS介导的放射敏化和生化调制之间的动力学关系。多种遗传和生化测试将确定通过HEDS治疗对KU氧化修饰的特异性敏感性。具体的目标2将确定HED的（生物）化学机制是细胞蛋白硫醇的氧化。具体目标3将研究DNA修复和DNA结构组织的其他方面，以确定它们对氧化还原调节的敏感性。