The Mechanism of Elimination of the Mitochondrial DNA Replisome

摘要

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.