Mechanisms of mitochondrial DNA replication licensing in trypanosomes

锥虫中线粒体 DNA 复制许可机制

• 批准号: 8431728
• 负责人: Ziyin Li
• 金额: $ 19万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8431728
• 关键词: 26S proteasome; Adopted; Affinity Chromatography; African; African Trypanosomiasis; Animals; Binding; Biological Assay; Catalytic Domain; Cell Cycle; Cells; Complex; Country; DNA; DNA biosynthesis; DNA copy number; Disease; Dissection; Drug Targeting; Ensure; Eukaryota; Eukaryotic Cell; Gene Silencing; Genome; Genome Stability; Goals; Guide RNA; Homologous Gene; Human; Infection; Kinetoplast DNA; Leishmania; Licensing; Licensing Factor; MCM2 gene; Maintenance; Mediating; Messenger RNA; Mitochondria; Mitochondrial DNA; Mitochondrial Proteins; Molecular; Monitor; Morphology; Names; Nuclear; Parasite Control; Parasites; Parasitic Diseases; Peptide Hydrolases; Pharmaceutical Preparations; Phase; Physiological; Proteins; RNA Interference; Recruitment Activity; Regulation; Regulatory Pathway; Replication Licensing; Replication Origin; Ribosomal RNA; Risk; Role; S Phase; Scheme; Shapes; Structure; System; Testing; Transcript; Trypanosoma; Trypanosoma brucei brucei; Trypanosomiasis; World Health Organization; chemotherapy; drug development; genetic regulatory protein; helicase; in vivo; novel; nuclear division; prevent; receptor; research study; segregation; vector

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.

描述（由申请人提供）：维持基因组稳定性需要准确的 DNA 复制。真核细胞通过将许可因子募集到复制起点来启动复制并随后通过 26S 蛋白酶体介导的许可因子降解以防止 DNA 重新复制来许可复制起点来实现这一点。真核生物还必须维持核外基因组线粒体 DNA (mtDNA) 的多个拷贝，但维持 mtDNA 拷贝数的根本机制仍然是个谜。目前的提案旨在了解 mtDNA 复制许可的分子机制，并建立在我们最近发现的 HslVU 蛋白酶作为布氏锥虫 (Trypanosoma brucei) 中 mtDNA 复制的重要调节因子的基础上。Trypanosoma brucei 是一种原生动物寄生虫，含有不寻常的 mtDNA 网络，称为动质体 DNA (kDNA)。这种蛋白酶是第一个已知的 kDNA 复制调节因子，也是在真核生物中鉴定出的第一个类似细菌的 HslVU 蛋白酶，但人们对其如何发挥其功能知之甚少，主要是因为其调节蛋白尚未鉴定。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，我们建议在本申请中检查它们在调节 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 TbHslVU 伴侣 1 和 2 的两种新蛋白（称为 VUP1 和 VUP2）充当 TbHslVU 的调节剂。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 调节蛋白的鉴定表明对 kDNA 复制的额外控制水平以及锥虫中 kDNA 复制调节方案的复杂性。最重要的是，由于在人类中没有发现 TbHslVU 及其伙伴 VUP1 和 VUP2 的同源物，因此它们是抗锥虫病化疗的潜在药物靶标。