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，描述通过氧化状态的变化和关键控制分子（通常是蛋白质）的还原来修饰功能。当细胞在一个被称为“氧化应激”的过程中受到活性氧的影响时，易受氧化还原调节的途径可能会改变。确定这种改变的机制通常是困难的，因为氧化应激还会对多种细胞靶标造成损伤，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对氧化还原调节蛋白的损伤与对细胞毒性靶标的损伤分离。我们的假设是DNA修复蛋白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，我们采用巯基乙醇的二硫化物，羟基-乙基二硫化物（HEDS）。正常细胞能够通过戊糖循环产生的还原当量来阻止蛋白质和非蛋白质硫醇与HEDS的硫-二硫交换，戊糖循环的关键调节酶是葡萄糖-6-磷酸脱氢酶（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，证实了上述效应的可逆性。通过该模型系统，我们将证明辐射致敏、DNA修复抑制和Ku与DNA末端结合的抑制都是由无毒浓度的HEDS孵育E89细胞引起的。这些效应在亲本细胞或A1A转染物中未见。正如“p53”可能是“基因组的守护者”，我们认为G6PD是“蛋白质的守护者”。这个应用程序将测试这个有趣的概念使用结合生化和遗传方法。特异性Aim 1将确定HEDS介导的放射致敏和生化调节之间的动力学关系。多种基因和生化试验将确定Ku对HEDS处理氧化修饰的特异性敏感性。特异性目的2将确定HEDS氧化细胞蛋白硫醇的（生物）化学机制。Specific Aim 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.