Studies on the organelle-specific functions of human Type 1A topoisomerase TOP3A

线粒体DNA缺失综合征是一组影响婴儿和儿童患者的临床异质性遗传疾病。已鉴定出与这些疾病相关的基因，然而，有相当数量的经证实的病例，其中负责该疾病的突变基因尚未被鉴定。本申请中提出的基础科学研究项目将大大提高我们对线粒体DNA缺失综合征分子机制的理解，并为患者的治疗开辟进一步的途径。在大多数生物体中，构建细胞体的生物材料都编码在DNA中。这种编码信息在称为RNA转录的过程中被读取。在细胞分裂之前，为了保持DNA含量在子细胞之间的均匀分布，DNA必须复制;这个过程称为DNA复制。在这些相当复杂的过程中，DNA必须打开，以便进入在DNA代谢中起作用的酶。解旋酶催化DNA的两条链的分离。在解旋酶解旋DNA双螺旋的过程中，产生了扭转应力，该应力由另一组称为拓扑异构酶的酶缓解。拓扑异构酶也能整理缠结的DNA片段，细胞的动力装置线粒体也含有DNA，但奇怪的是，这种DNA和大多数细菌细胞的DNA一样是环状的，而不像真核生物细胞核中的线性染色体DNA。线粒体DNA比核DNA更容易受到损伤，因为线粒体中发生的代谢过程。保护细胞核和线粒体DNA完整性的酶之一是DNA拓扑异构酶III α（TOP 3A）。在小鼠中完全缺乏这种酶会导致早期胚胎发育期间的死亡，而其在细胞培养中的水平降低会导致细胞加速老化。虽然TOP 3A的生物化学活性已经得到了很好的表征，但其确切功能在很大程度上是未知的，关于其在维持线粒体基因组完整性方面的作用更是知之甚少。在我的初步工作中，我已经建立了一个系统，该系统允许从细胞中耗尽TOP 3A，而特异性表达和功能可以仅在细胞核或线粒体中维持。该系统允许对TOP 3A的核和线粒体功能进行相互独立的分析，以揭示为什么在缺乏TOP 3A的细胞中加速衰老过程，以及它是否可以归因于对细胞核或线粒体特异性的酶的功能。更具体地说，我们将研究线粒体DNA的复制和线粒体的代谢活性如何在缺乏线粒体TOP 3A功能的细胞中受到影响。我们将研究抑制核TOP 3A功能如何影响核DNA代谢，染色体分离和细胞分裂。我们还将鉴定和鉴定有助于其细胞器特异性功能的TOP 3A蛋白伙伴。

Mitochondrial DNA depletion syndromes are a group of clinically heterogeneous genetic diseases that affect patients in infancy and childhood. Hitherto nine genes have been identified to be associated to these diseases, however, there are a considerable number of verified cases, where the mutated gene responsible for the disease has not been identified. The basic science research project proposed in this application will considerably enhance our understanding of the molecular mechanisms responsible for mitochondrial DNA depletion syndromes, and open up further avenues for the treatment of its sufferers.In most living organisms biomaterial that builds the body of cells is coded in the DNA. This coded information is read in a process termed RNA transcription. Before the division of cells, in order to maintain the equal distribution of DNA content between the daughter cells, the DNA has to be duplicated; this process is termed DNA replication. During these rather complicated processes, the DNA has to open up to give access to enzymes that function in DNA metabolism. Separation of the two strands of DNA is catalysed by helicase enzymes. During the unwinding of the DNA double helix by the helicase enzymes, torsional stress is generated, which is relieved by another set of enzymes called topoisomerases. Topoisomerases are also capable of tidying up tangled stretches of DNA.The powerplant of cells, the mitochondria, also contain DNA, but - curiously enough - this DNA is circular like DNA of most bacterial cells, and unlike the linear chromosomal DNA that can be found in the nucleus of eukaryotes. Mitochondrial DNA is more susceptible to damage than nuclear DNA because of the metabolic processes that take place in the mitochondria. One of the enzymes that guard the integrity of nuclear as well as mitochondrial DNA is DNA topoisomerase III alpha (TOP3A). The complete lack of this enzyme in mice causes lethality during early embryonic development, while its reduced levels in cell culture cause the accelerated ageing of cells. Though the biochemical activities of TOP3A have been well characterised, its exact function is largely unknown, and even less is known about its role in maintaining the integrity of the mitochondrial genome. In my preliminary work I have set up a system that allows the depletion of TOP3A from the cells, while specific expression and function can be maintained in either the nuclei or in the mitochondria, exclusively. This system permits the analysis of nuclear and mitochondrial functions of TOP3A independently of each other to reveal why the ageing process is accelerated in cells devoid of TOP3A, and whether it can be attributed to a function of the enzyme specific to the nuclei or the mitochondria. More specifically, we will investigate how the replication of mitochondrial DNA, and the metabolic activity of mitochondria are affected in cells that lack mitochondrial TOP3A function. We will study how suppression of nuclear TOP3A functions affects nuclear DNA metabolism, chromosome segregation and cell divisions. We will also identify and characterise protein partners of TOP3A that assist its organelle-specific functions.