The Mechanism of Elimination of the Mitochondrial DNA Replisome

摘要 人类线粒体DNA(MtDNA)复制过程中的缺陷导致 引起多种退行性疾病的突变，涉及多个器官和系统 人体。在这个项目中，我们建议研究一种可能的机制，以防止形成 线粒体DNA的大规模缺失，这是线粒体基因组最常见的(从头)缺陷。 缺失的形成机制尚不清楚，但迄今报道的研究表明，它们起源于 线粒体DNA复制机制(复制体)停滞，导致DNA断裂 思特斯。值得注意的是，显著的mtdna复制停滞位点与主要的 线粒体蛋白水解酶，Lon，这表明它可能参与消除停滞的复制体。在……里面 此外，已发现人类线粒体Hsp70/40伴侣系统的组件共沉淀 与线粒体复制聚合酶的催化亚单位有关，这表明这些可能与 在线粒体复制体的组装或拆卸中。事实上，在模式生物中，Hsp70/40系统具有 已被发现通过展开和运送蛋白质底物来辅助Lon蛋白酶。另一方面，Hsp70/40 系统还被发现与另一种蛋白酶ClpXP协同作用，这一缺陷导致 线粒体基因组不稳定。 这项建议旨在评估停滞的线粒体DNA复制体被两个 使用Lon或ClpXP蛋白酶的替代机制。此外，Hsp70/40分子伴侣系统 可用于分解复制体并将其组分传递给客户的蛋白酶。确认和 细胞去除有缺陷线粒体的能力之间的直接关系的特征 复制体和线粒体基因组中积累的损伤，将带来一种新颖而令人兴奋的 线粒体DNA缺失相关人类疾病的发展前景，潜在地确定了 前瞻性治疗策略。为此，我们将采取综合方法，将切割- 分析分子亲和力和动力学参数的生物分子层干涉法边缘技术 需要专门的酶分析的方法性生化分析，并测试假定的相关性 Lon、Hsp70/40和ClpX的细胞水平与体内mtDNA缺失的发展之间的关系 以酿酒酵母为模式生物。到目前为止，我们已经证明了Lon蛋白酶水解酶 线粒体复制聚合酶的催化亚基，但仅在没有剩余亚基的情况下 聚合酶亚基。这一发现证实了我们最初的假设，并证明了研究 HSP70/40和/或ClpX在复制体复合体的解体中的作用，以使催化核心降解 作者：Lon Protease。

Abstract Defects in the human mitochondrial DNA (mtDNA) replication process result in the accumulation of mutations that give rise to a broad spectrum of degenerative disorders involving multiple organs and systems of the human body. In this project, we propose to investigate a putative mechanism that prevents the formation of large-scale deletions in mtDNA, which are the most common (de novo) defects of the mitochondrial genome. The mechanism of deletion formation is unknown, but studies reported to date indicate that they originate from stalling of the mitochondrial DNA replication machinery (the replisome), which leads to the breaking of DNA strands. Notably, prominent mtDNA replication stalling sites correspond with binding sites of the major mitochondrial protease, Lon, which suggests that it might be involved in the elimination of halted replisomes. In addition, the components of human mitochondrial Hsp70/40 chaperone system have been found to co-precipitate with the catalytic subunit of the mitochondrial replicative polymerase, which suggests that these may be involved in the assembly or disassembly of mitochondrial replisomes. In fact, in model organisms, Hsp70/40 systems have been found to assist Lon protease by unfolding and delivering protein substrates. On the other hand, Hsp70/40 systems have also been found to cooperate with another protease, ClpXP, which deficiency results in mitochondrial genome destabilization. This proposal aims to evaluate the hypothesis that stalled mtDNA replisomes are eliminated by two alternative mechanisms that engage either Lon or ClpXP protease. In addition, the Hsp70/40 chaperone system may serve to disassemble the replisome and deliver its components to the client protease. Confirmation and characterization of a direct relationship between the capacity of a cell to remove defective mitochondrial replisomes and the accumulation of damage in the mitochondrial genome, would bring a novel and exciting perspective on the development of mtDNA deletions-linked human disorders, potentially identifying targets for prospective therapeutic strategies. To this end, we will apply a comprehensive approach combining the cutting- edge technique of biolayer interferometry for the analysis of molecular affinities and kinetic parameters, a methodical biochemical analysis that entails specialized enzymatic assays, and testing the putative correlation between cellular levels of Lon, Hsp70/40 and ClpX and the development of mtDNA deletions in vivo, using Saccharomyces cerevisiae as the model organism. To date, we have demonstrated that Lon protease hydrolases the catalytic subunit of the mitochondrial replicative polymerase, but only in the absence of the remaining polymerase subunits. This finding confirms our initial hypothesis and warrants the investigation of the role of Hsp70/40 and/or ClpX in the disassembly of the replisome complex to enable degradation of the catalytic core by Lon protease.