The mechanism of elimination of the mitochondrial DNA replisome - 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 C
