Mechanisms of mitochondrial DNA replication licensing in trypanosomes

描述（由申请人提供）：基因组稳定性的维持需要准确的DNA复制。真核细胞通过将许可因子募集到复制起点以启动复制，随后通过26S蛋白酶体介导的许可因子降解以防止DNA再复制，从而许可复制起点来实现这一点。真核生物也必须维持一个多核基因组的多个拷贝，即线粒体DNA（mtDNA），但维持mtDNA拷贝数的潜在机制仍然是一个谜。目前的建议是旨在了解mtDNA复制许可的分子机制，并建立在我们最近发现的HslVU蛋白酶作为一个重要的调节剂的mtDNA复制在布氏锥虫，一种原生动物寄生虫，包含一个不寻常的mtDNA网络，称为动基体DNA（kDNA）。这种蛋白酶是第一个已知的kDNA复制调节剂和第一个在真核生物中鉴定的细菌样HslVU蛋白酶，但它如何发挥其功能的了解很少，主要是因为它的调节蛋白没有被鉴定。通过串联亲和纯化，我们鉴定了两种在体内与TbHslVU相关的新型线粒体蛋白，并且我们在本申请中提出检查它们在调节TbHslVU中的潜在作用以及它们在kDNA复制中的潜在参与。我们假设两种新的蛋白质，命名为VUP1和VUP2的TbHslVU伴侣1和2，作为调节剂的TbHslVU的功能。它们可以激活或抑制TbHslVU的活性，或调节TbHslVU复合物的组装，或介导底物识别。TbHslVU的调节蛋白的鉴定表明对kDNA复制的额外水平的控制和锥虫中kDNA复制的调节方案的复杂性。最重要的是，由于在人类中没有发现TbHslVU及其配偶体VUP1和VUP2的同源物，因此它们是抗锥虫化疗的潜在药物靶标。

DESCRIPTION (provided by applicant): Maintenance of genomic stability requires accurate DNA replication. Eukaryotic cells achieve this by licensing the replication origins through recruiting the licensing factors to the origins to initiate replication and subsequently by 26S proteasome-mediated degradation of the licensing actors to prevent DNA re-replication. Eukaryotes must also maintain multiple copies of an extranuclear genome, the mitochondrial DNA (mtDNA), but the underlying mechanism for maintenance of mtDNA copy number remains a mystery. The current proposal is aimed at understanding the molecular mechanism of mtDNA replication licensing and is built on our recent discovery of HslVU protease as an essential regulator of mtDNA replication in Trypanosoma brucei, a protozoan parasite that contains an unusual mtDNA network, known as the kinetoplast DNA (kDNA). This protease is the first known regulator of kDNA replication and first bacterial-like HslVU protease identified in a eukaryote, but how it exerts its function is poorly understood, mainly because its regulatory proteins are not identified. Through tandem affinity purification, we identified two novel mitochondrial proteins that associate with TbHslVU in vivo, and we propose in this application to examine their potential roles in regulating TbHslVU as well as their potential involvement in kDNA replication. We hypothesize that the two novel proteins, named VUP1 and VUP2 for TbHslVU Partner 1 and 2, function as regulators of TbHslVU. They could either activate or inhibit the activity of TbHslVU or regulate the assembly of TbHslVU complex or mediate substrate recognition. The identification of regulatory proteins of TbHslVU suggests an additional level of control over the replication of kDNA and the complexity of the regulatory scheme of kDNA replication in trypanosomes. Most importantly, since no homologs of TbHslVU and its partners, VUP1 and VUP2, are found in humans, they are potential drug targets for anti-trypanosomiasis chemotherapy.