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

线粒体DNA缺失综合征是一组临床异质性遗传病，影响患者在婴儿期和儿童期。到目前为止，已经确定有9种基因与这些疾病有关，但是，有相当多的经证实的病例，其中导致疾病的突变基因尚未确定。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耗竭综合征分子机制的理解，并为其患者的治疗开辟进一步的途径。在大多数生物体中，构成细胞体的生物材料被编码在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，为了在子细胞之间保持DNA含量的均匀分布，DNA必须被复制；这个过程被称为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。这两条DNA链的分离是由解旋酶催化的。在解旋酶解DNA双螺旋的过程中，会产生扭转应力，扭转应力由另一组被称为拓扑异构酶的酶来缓解。拓扑异构酶还能整理DNA的纠缠片段。细胞的动力装置线粒体也含有DNA，但奇怪的是，这种DNA像大多数细菌细胞的DNA一样是圆形的，而不像真核生物细胞核中发现的线状染色体DNA。线粒体DNA比核DNA更容易受到损伤，因为线粒体中发生了代谢过程。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 (TOP3A)。在小鼠中完全缺乏这种酶会导致胚胎早期发育过程中的死亡，而在细胞培养中，这种酶的水平降低会导致细胞加速衰老。尽管对TOP3A的生化活性已经有了很好的描述，但其确切的功能在很大程度上是未知的，对其在维持线粒体基因组完整性方面的作用更是知之甚少。在我的前期工作中，我已经建立了一个系统，可以使TOP3A从细胞中耗尽，而特定的表达和功能可以只在细胞核或线粒体中维持。该系统允许对细胞核和线粒体的TOP3A功能进行相互独立的分析，以揭示为什么在缺乏TOP3A的细胞中衰老过程会加速，以及它是否可以归因于细胞核或线粒体特异性酶的功能。更具体地说，我们将研究缺乏线粒体TOP3A功能的细胞如何影响线粒体DNA的复制和线粒体的代谢活性。我们将研究抑制核TOP3A功能如何影响核DNA代谢、染色体分离和细胞分裂。我们还将鉴定和表征TOP3A的蛋白质伙伴，协助其细胞器特异性功能。

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.