The Mechanism of Elimination of the Mitochondrial DNA Replisome

线粒体DNA复制体的消除机制。 线粒体是真核细胞的重要细胞器，它从食物中转化化学能。 转化为三磷酸腺苷（ATP）的磷酸酐键。人类线粒体基因组 编码对ATP合成至关重要的蛋白质，因此，线粒体DNA（mtDNA）的维持缺陷 导致能量剥夺并可能导致涉及心脏的退行性疾病的发展， 肌肉、肾脏、肝脏和中枢神经系统（1-3）。例如，Alpers综合征的特征在于： 顽固性癫痫、精神发育迟滞和肝功能衰竭，导致儿童早期死亡（4、5）。缺陷 线粒体DNA的维持也与其他突出的疾病有关，如帕金森病和阿尔茨海默病 疾病，自闭症谱系障碍，糖尿病，以及多种类型的癌症和衰老（6-13）。的 线粒体疾病的发病机制尚不清楚。任何线粒体DNA都无法治愈- 相关疾病，目前只有姑息治疗策略可用（14）。 PI建议研究防止大规模缺失形成的假定机制 在线粒体DNA中，这是线粒体基因组最常见的（从头）缺陷（15-17）。机制 缺失的形成是未知的，但迄今为止的研究表明，它们通常起源于mtDNA 复制停滞，这促进了DNA链的断裂。缺失最有可能在以下过程中形成： DNA断裂修复（18-20）。值得注意的是，缺乏特定的线粒体分子伴侣和蛋白酶， 促进mtDNA的不稳定和缺失的积累（21-27），这意味着它们在 防止缺失形成。另一方面，我们的初步结果表明，一个停滞的线粒体 复制聚合酶在很长一段时间内保持与DNA结合，这可能是有害的，并且可能 需要主动消除。因此，我们推断，在正常情况下，功能失调的mtDNA复制体是 通过特定的伴侣蛋白和蛋白酶消除，这反过来又促进复制重新启动。病理 在这种条件下，复制停滞的频率增加（例如由于复制酶的缺陷）超过了 假定的消除系统的能力导致DNA断裂频率的增加， 有害的修复机制（我们在最近的综述中详细讨论了这一点（20））。值得注意的是， 观察到随着年龄的增长，大规模的缺失在组织中积累（12，13，28），奇怪的是， 已经观察到相关的伴侣蛋白和蛋白酶也随着年龄的增长而减少（29-31）。这种明显 相关性要求调查因果关系。此外，假定的关系 分子伴侣/蛋白酶系统和缺失承载（Δ）mtDNA分子的积累最近获得 重要的认识，由于报告表明mtUPR（未折叠蛋白质反应）需要快速 mtDNA群体中ΔmtDNA的扩增，这反过来又加剧了相关疾病的发展 (32，33）。ΔmtDNA克隆性扩增的分子基础仍不清楚。明显地，蛋白质和 我们提出要研究的机制似乎是ΔmtDNA克隆扩张的核心。总的来说， 该项目将有助于理解ΔmtDNA形成及其克隆扩张的机制， 目前，该领域的主要挑战。此外，直接关系的确定和定性 细胞去除有缺陷的线粒体复制体的能力和线粒体复制体的完整性之间的关系 基因组将为该领域带来一个关于线粒体疾病发展的新的和令人兴奋的视角， 具有治疗应用的潜力。 目的：阐明人Lon和ClpXP蛋白酶以及Hsp 70/Hsp 40分子伴侣系统在细胞凋亡中的作用 核心线粒体DNA复制体的消除。 线粒体DNA复制体的主要停滞位点（34，35）与线粒体DNA的主要结合位点相对应。 线粒体蛋白酶，Lon（36）。以前对各种模式生物的研究表明，Lon经常 需要分子伴侣Hsp 70/40系统的帮助，该系统展开并递送蛋白质底物（37）。 Hsp 70/40系统还可以与另一种线粒体蛋白酶ClpXP协作（38-40）。失去了Lon， Hsp 40和ClpX损害体内mtDNA的稳定性（21，24，27）。因此，PI提出，停滞的mtDNA 复制体通过两种替代机制消除，这两种机制涉及Lon或ClpXP蛋白酶。此外，本发明还提供了一种方法， Hsp 70/40分子伴侣系统可用于分解复制体并将其组分递送至细胞。 客户蛋白酶（图1）。我们将通过应用综合方法来评估这一假设， 生物层干涉测量的尖端技术，用于分析分子亲和力和动力学 参数，一个有条不紊的生化分析，需要专门的酶测定，并测试是否 升高的Lon、Hsp 70/40和ClpX水平可以减轻体内诱导缺失的形成， 酿酒酵母作为模型。

The mechanism of elimination of the mitochondrial DNA replisome. Specific Aims: Mitochondria are essential organelles of eukaryotic cells that convert chemical energy from food into that of the phosphoanhydride bonds of adenosine triphosphate (ATP). The human mitochondrial genome encodes proteins critical for ATP synthesis, therefore, defects in the maintenance of mitochondrial DNA (mtDNA) result in energy deprivation and may lead to the development of degenerative disorders involving the heart, muscles, kidneys, liver and the central nervous system (1-3). For example, Alpers syndrome is characterized by intractable epilepsy, psychomotor retardation and liver failure that leads to death in early childhood (4,5). Defects of mtDNA maintenance have also been linked to other prominent disorders such as Parkinson’s and Alzheimer’s diseases, autism spectrum disorders, diabetes, as well as multiple types of cancer and aging (6-13). The mechanisms of pathogenesis of mitochondrial diseases are unknown. There is no cure for any of the mtDNA- associated diseases and only palliative treatment strategies are currently available (14). The PI proposes 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 (15-17). The mechanism of deletions formation is unknown, but studies reported to date indicate that they commonly originate from mtDNA replication stalling, which promotes breakage of DNA strands. Deletions are most likely formed in the process of DNA breaks repair (18-20). Notably, the absence of specific mitochondrial molecular chaperones and proteases promotes the destabilization of mtDNA and accumulation of deletions (21-27), which implies their role in preventing deletions formation. On the other hand, our preliminary results indicate that a stalled mitochondrial replicative polymerase remains DNA-bound for a significant extent of time, which could be deleterious and likely requires active elimination. Therefore, we infer that, in normal conditions, dysfunctional mtDNA replisomes are eliminated by specific chaperones and proteases, which in turn promotes replication restart. In pathological conditions, the increased frequency of replication stalling (e.g. due to defects of the replicative enzymes) exceeds the capacity of the putative elimination system resulting in an increase in DNA breaks frequency and the initiation of the deleterious repair mechanism (we discussed this in detail in a recent review (20)). Notably, it has been observed that the large-scale deletions accumulate in tissues with age (12,13,28) and, curiously, the activity of the related chaperones and proteases has been observed to decrease with age as well (29-31). This apparent correlation calls for the investigation of a causative relationship. In addition, the putative relationship between chaperones/proteases systems and the accumulation of deletion-bearing (Δ)mtDNA molecules recently gained significant recognition, due to reports indicating that mtUPR (unfolded protein response) warrants rapid expansion of ΔmtDNA in the mtDNA population, which in turn exacerbates the development of related disorders (32,33). The molecular basis of the clonal expansion of ΔmtDNA remains elusive. Markedly, the proteins and mechanisms that we propose to investigate appear to be central to the clonal expansion of ΔmtDNA. In summary, the project will help to understand the mechanism of ΔmtDNA formation and their clonal expansion, which are currently the major challenges in the field. Furthermore, identification and characterization of a direct relationship between the capacity of a cell to remove defective mitochondrial replisomes and the integrity of the mitochondrial genome would bring to the field a novel and exciting perspective on the development of mitochondrial disorders, with a potential for therapeutic applications. Aim: To elucidate the role of human Lon and ClpXP proteases, and the Hsp70/Hsp40 chaperone system in the elimination of the core mitochondrial DNA replisome. Prominent mtDNA replisome stalling sites (34,35) correspond with binding sites of the major mitochondrial protease, Lon (36). Previous studies from various model organisms indicated that Lon often requires the assistance of a chaperone Hsp70/40 system, which unfolds and delivers protein substrates (37). The Hsp70/40 system can also cooperate with another mitochondrial protease, ClpXP (38-40). Loss of Lon, Hsp40 and ClpX impairs mtDNA stability in vivo (21,24,27). Therefore, the PI proposes that the stalled mtDNA replisome is 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 (Figure 1). We will evaluate this hypothesis by applying a comprehensive approach combining the cutting-edge technique of biolayer interferometry for the analysis of molecular affinities and kinetic parameters, a methodical biochemical analysis entailing specialized enzymatic assays, and testing whether elevated levels of Lon, Hsp70/40 and ClpX can alleviate the formation of induced deletions in vivo using Saccharomyces cerevisiae as a model.