Oxidative Damage to DNA Repair Pathways

描述（由申请人提供）：氧化还原调节是一个术语，描述了通过关键控制分子（通常是蛋白质）的氧化与还原状态的变化来改变功能。当细胞在被描述为“氧化应激”的过程中受到活性氧物质的影响时，对氧化还原调节敏感的途径可能会改变。定义这种改变的机制通常是困难的，因为氧化应激可以另外引起对多个细胞靶点的损伤，包括膜、细胞器和染色质，通常导致有丝分裂或凋亡死亡。我们提出的实验将允许从对细胞毒性靶点的损伤中分离对氧化还原调节蛋白的损伤。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温和和特异性氧化，我们使用巯基乙醇的二硫化物，羟乙基二硫化物（HEDS）。正常细胞能够通过还原戊糖循环产生的当量来防止其蛋白质和非蛋白质硫醇与HEDS的硫醇-二硫键交换，戊糖循环的关键调节酶是葡萄糖-6-磷酸脱氢酶（G6 PD）。为了防止这种情况，我们研究了没有G6 PD活性的CHO细胞系（E89）。通过将G6 PD基因转移回E89突变体中，确定了观察到的效应的可逆性。利用该模型系统，我们将证明辐射敏化、DNA修复的抑制和Ku与DNA末端结合的抑制都是由E89细胞与无毒浓度的HEDS孵育引起的。这些效应在亲本细胞或A1 A转染子中未观察到。正如“p53”可能是“基因组的守护者”一样，我们认为G6 PD是“蛋白质的保护者”。本申请将使用生物化学和遗传学相结合的方法来测试这个有趣的概念。具体目标1将确定HEDS介导的放射增敏和生化调节之间的动力学关系。多种遗传和生化测试将确定Ku对HEDS处理的氧化修饰的特异性敏感性。具体目标2将确定HEDS氧化细胞蛋白硫醇的（生物）化学机制。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。

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.