Oxidative stress and metabolic reconfiguration in the development of diseases with underlying deficiencies in DNA repair.

我们以前的研究表明，由于未修复的DNA损伤导致的聚(ADP-核糖)聚合酶过度激活导致的NAD+的枯竭，对细胞的氧化还原状态有深远的影响，从而导致活性氧物种(ROS)水平的升高。ROS的主要和最相关的靶点之一是DNA，导致DNA损伤，然后可能被修复为突变。无法有效处理DNA损伤的细胞，例如那些DNA修复酶潜在缺陷的细胞，似乎面临着双重挑战：最初的遗传毒性侮辱和随之而来的氧化应激。这项建议的目的之一是研究多聚ADP-核糖聚合酶过度激活和氧化应激在缺乏修复DNA双链断裂的关键角色的疾病中的作用：ATM、NBN、XLF、LIGIV、FANCD1/BRCA2、FANCN/PALB2等。由DNA损伤引起的永久性应激的影响通常通过所谓的代谢重构来补偿。例如，DNA损伤后，激活的ATM通过葡萄糖-6-磷酸脱氢酶促进磷酸戊糖途径，从而增加NADPH的产生和提高抗氧化能力。在这个项目中，我们将研究一组不同但重叠的细胞系，这些细胞系在修复DNA双链断裂方面存在缺陷，以寻找由抗氧化反应元件(ARE)调节的基因变化，期望ROS作为正常的内源性代谢物出现，例如在线粒体呼吸期间，可能是代谢重组的目标。Ares的多态也将被检测，特别是关于DNA修复缺陷奈梅根断裂综合征(NBS)的临床变异。为此，我们从临床特征良好的患者中提取了40多个细胞系，它们都具有相同的纯合子创始人突变。对于这些细胞系中的许多细胞，已经确定了NBN蛋白亚型片段的数量，并与临床病程，特别是癌症发生率相关。

Our own previous studies indicate that depletion of NAD+ due to hyperactivation of poly(ADP-ribose) polymerase, as a consequence of unrepaired DNA damage, has a profound effect on the redox status of a cell leading to raised levels of reactive oxygen species (ROS). One of the main and most relevant targets of ROS is DNA, leading to DNA damage which may then be fixed as mutations. Cells unable to deal efficiently with DNA damage, such as those with an underlying deficiency in DNA repair enzymes, seemingly have a double challenge: the primary genotoxic insult and the ensuing oxidative stress. One aim of this proposal is to examine the role of poly(ADP-ribose) polymerase hyperactivation and oxidative stress in diseases which are deficient in key players in the repair of DNA double-strand breaks: ATM, NBN, XLF, LIGIV, FANCD1/BRCA2, FANCN/PALB2 etc. The effect of permanent stress due to DNA damage is often compensated for by so called metabolic reconfiguration. For example, after DNA damage, activated ATM promotes the pentose phosphate pathway via glucose-6-phosphate dehydrogenase leading to increased NADPH production and improved antioxidant capacity. In this project we will examine a collection of cell lines from distinct, but overlapping, disorders with deficiencies in the repair of DNA double-strand breaks for changes in genes regulated by antioxidant response elements (ARE) in the expectation that ROS arising as normal endogenous metabolites, for example during mitochondrial respiration, might be the target of metabolic reconfiguration. Polymorphisms in AREs will also be examined, particularly with respect to clinical variation in the DNA repair deficiency Nijmegen Breakage Syndrome (NBS). For this we have over 40 cell lines from clinically well characterised patients, all with the same homozygous founder mutation. For many of these cell lines, the amount of hypomorphic NBN protein fragment has been determined and correlated with clinical course, particularly cancer incidence.