DNA Topoisomerase Misfunctioning in Neurological Disease

DNA拓扑异构酶II(TOP2)对有效转录所需的DNA结构进行改变， 复制和DNA修复。这种酶在DNA中引入瞬间双链断裂，通过 蛋白质/DNA共价中间体称为裂解复合体。TOP2的DNA切割机制 允许细胞催化DNA构象的变化，而不会有坦率的DNA双链断裂的危险。 哺乳动物细胞含有两种TOP2亚型，分别称为TOP2α和TOP2β。这两种酶有截然不同的 TOP2β的生物学功能在转录和染色体结构中具有独特的作用。Top2β为 对于神经元基因的转录尤其重要。有趣的是，拓扑异构酶最近被 在长基因的转录中特别重要，提示拓扑异构酶 在神经细胞中，功能可能是唯一重要的。而TOP2催化机制通常避免 产生双链断裂，在某些情况下，有人认为TOP2β可以持久存在 在转录调控起始过程中，双链断裂。最近的结果也 提示长时间的拓扑异构酶共价复合体可能导致神经毒性DNA损伤。二 最近的报道发现，在自闭症患者中存在相同的TOP2杂合β突变 精神障碍(ASD)。在初步数据中，我们发现在两个独立的自闭症患者中发现了TOP2β突变 患者通过产生酶介导的DNA链产生自发的DNA损伤 休息一下。在这一应用中，我们建议探索这类酶的生化特性 体外酶分析及突变酶在酵母和哺乳动物细胞中的表达。第二个目标是 我们的研究是研究DNA对神经细胞中TOP2β蛋白的损伤作用。我们的合作者彼得·麦金农来自圣彼得堡。 裘德儿童医院培养的小鼠胚胎干细胞在体外表达两种不同的TOP2β突变 导致拓扑异构酶介导的切割升高。我们计划描述DNA损伤反应和 当这些细胞分化为神经元谱系时的发育缺陷。最后，我们建议发起 人神经母细胞瘤SH-SY5Y细胞导入拓扑异构酶模型系统的建立 能够对人类神经细胞产生自发DNA损伤的突变。我们将决定 TOP2β的DNA损伤突变是否与功能突变的丧失有类似的影响，或者 拓扑异构酶诱导DNA损伤的产生对神经细胞分化和 生死存亡。这些研究将强调内源性DNA损伤在人类神经学中的潜在作用 并提供了一个模型系统，可以用来探索拓扑异构酶错配的独特方式。 功能正常可能会导致人类疾病。

DNA topoisomerase II (Top2) carries out changes in DNA structure needed for efficient transcription, replication, and DNA repair. This enzymes introduce transient double strand breaks in DNA through a protein/DNA covalent intermediate termed the cleavage complex. The DNA cleavage mechanism of Top2 allows cells to catalyze changes in DNA conformation without the dangers of frank DNA double strand breaks. Mammalian cells contain two Top2 isoforms termed Top2α and Top2β. The two enzymes have distinct biological functions with Top2β having unique roles in transcription and chromosome structure. Top2β is particularly important for transcription of neuronal genes. Interestingly, topoisomerases have recently been suggested to be particularly important in the transcription of long genes, suggesting that topoisomerase function may be uniquely important in neuronal cells. While the Top2 catalytic mechanism typically avoids generating double strand breaks, in some contexts, it has been suggested that Top2β makes long lasting double strand breaks during the process of regulated transcription initiation. Recent results have also suggested that long lasting topoisomerase covalent complexes may cause neurotoxic DNA damage. Two recent reports have identified identical heterozygous Top2β mutations in patients with autism spectrum disorders (ASDs). In preliminary data, we show that the Top2β mutation found in the two independent ASD patients generates spontaneous DNA damage through the generation of enzyme mediated DNA strand breaks. In this application, we propose to explore the biochemical characteristics of this type of enzyme using in vitro enzyme assays and expression of the mutant enzyme in yeast and mammalian cells. A second aim of our studies is to study DNA damaging Top2β proteins in neuronal cells. Our collaborator Peter McKinnon, St. Jude Children's Hospital, has generated mouse ES cells that express two distinct Top2β mutations that in vitro lead to elevated topoisomerase mediated cleavage. We plan to characterize DNA damage responses and developmental defects when these cells differentiate into neuronal lineages. Finally, we propose to initiate development of a model system using human neuroblastoma SH-SY5Y cells to introduce topoisomerase mutations capable of generating spontaneous DNA damage into human neuronal cells. We will determine whether DNA damaging mutations of Top2β have similar effects to loss of function mutations, or whether the generation of topoisomerase induced DNA damage leads to unique effects on neuronal cell differentiation and survival. These studies will highlight potential roles of endogenous DNA damage in human neurological diseases, and provide a model system that can be used to explore unique ways that topoisomerase mis- functioning can contribute to human diseases.