Gotowa bibliografia na temat „MtDNA Replisome”

The unit of inheritance for mitochondrial DNA (mtDNA) is a complex nucleoprotein structure termed the nucleoid. The organization of the nucleoid as well as its role in mtDNA replication remain largely unknown. Here, we show in Saccharomyces cerevisiae that at least two populations of nucleoids exist within the same mitochondrion and can be distinguished by their association with a discrete proteinaceous structure that spans the outer and inner mitochondrial membranes. Surprisingly, this two membrane–spanning structure (TMS) persists and self-replicates in the absence of mtDNA. We tested whether TMS functions to direct the replication of mtDNA. By monitoring BrdU incorporation, we observed that actively replicating nucleoids are associated exclusively with TMS. Consistent with TMS's role in mtDNA replication, we found that Mip1, the mtDNA polymerase, is also a stable component of TMS. Taken together, our observations reveal the existence of an autonomous two membrane–spanning mitochondrial replisome as well as provide a mechanism for how mtDNA replication and inheritance may be physically linked.

Each nucleated cell contains several hundreds of mitochondria, which are unique organelles in being under dual genome control. The mitochondria contain their own DNA, the mtDNA, but most of mitochondrial proteins are encoded by nuclear genes, including all the proteins required for replication, transcription, and repair of mtDNA. MtDNA replication is a continuous process that requires coordinated action of several enzymes that are part of the mtDNA replisome. It also requires constant supply of deoxyribonucleotide triphosphates(dNTPs) and interaction with other mitochondria for mixing and unifying the mitochondrial compartment. MtDNA maintenance defects are a growing list of disorders caused by defects in nuclear genes involved in different aspects of mtDNA replication. As a result of defects in these genes, mtDNA depletion and/or multiple mtDNA deletions develop in affected tissues resulting in variable manifestations that range from adult-onset mild disease to lethal presentation early in life.

Human mitochondrial DNA (mtDNA) is a 16.9 kbp double-stranded, circular DNA, encoding subunits of the oxidative phosphorylation electron transfer chain and essential RNAs for mitochondrial protein translation. The minimal human mtDNA replisome is composed of the DNA helicase Twinkle, DNA polymerase γ, and mitochondrial single-stranded DNA-binding protein. While the mitochondrial RNA transcription is carried out by mitochondrial RNA polymerase, mitochondrial transcription factors TFAM and TFB2M, and a transcription elongation factor, TEFM, both RNA transcriptions, and DNA replication machineries are intertwined and control mtDNA copy numbers, cellular energy supplies, and cellular metabolism. In this review, we discuss the mechanisms governing these main pathways and the mtDNA diseases that arise from mutations in transcription and replication machineries from a structural point of view. We also address the adverse effect of antiviral drugs mediated by mitochondrial DNA and RNA polymerases as well as possible structural approaches to develop nucleoside reverse transcriptase inhibitor and ribonucleosides analogs with reduced toxicity.

Faithful replication of the mitochondrial genome is carried out by a set of key nuclear-encoded proteins. DNA polymerase γ is a core component of the mtDNA replisome and the only replicative DNA polymerase localized to mitochondria. The asynchronous mechanism of mtDNA replication predicts that the replication machinery encounters dsDNA and unique physical barriers such as structured genes, G-quadruplexes, and other obstacles. In vitro experiments here provide evidence that the polymerase γ heterotrimer is well-adapted to efficiently synthesize DNA, despite the presence of many naturally occurring roadblocks. However, we identified a specific G-quadruplex–forming sequence at the heavy-strand promoter (HSP1) that has the potential to cause significant stalling of mtDNA replication. Furthermore, this structured region of DNA corresponds to the break site for a large (3,895 bp) deletion observed in mitochondrial disease patients. The presence of this deletion in humans correlates with UV exposure, and we have found that efficiency of polymerase γ DNA synthesis is reduced after this quadruplex is exposed to UV in vitro.

There is a worldwide trend for women to have their first pregnancy later in life. However, as oocyte quality declines with maternal aging, this trend leads to an increase in subfertility. The cellular mechanisms underlying this decline in oocyte competence are poorly understood. Oocyte mitochondria are the subcellular organelles that supply the energy that drives early embryogenesis, and thus their quality is critical for successful conception. Mitochondria contain their own DNA (mtDNA) and mutations in mtDNA cause mitochondrial diseases with severe symptoms, such as neurodegeneration and heart disease. Since mitochondrial function declines in tissues as humans age accompanied by an accumulation of mtDNA mutations, mtDNA is implicated as a cause of declining oocyte quality in older mothers. While this mutation load could be caused by declining accuracy of the mitochondrial replisome, age-related decline in mitochondrial quality control likely contributes, however knowledge is lacking. Mitophagy, a cellular process which specifically targets and recycles damaged mitochondria may be involved, but studies are scarce. And although assisted reproductive technologies can help older mothers, how these techniques affect the mechanisms that regulate mitochondrial and oocyte quality have not been studied. With the long-term goal of understanding the molecular mechanisms that control mitochondrial quality in the oocyte, model systems including Drosophila and mouse as well as human oocytes have been used. In this review, we explore the contribution of mitophagy to oocyte quality and the need for further systematic investigation in oocytes during maternal aging using different systems. Lay summary Mitochondria are small parts of cells called organelles that generate the chemical energy needed for life. Hundreds of thousands of mitochondria in the developing eggs of the mother support the initial growth and development of the fertilized egg. However, due to increasingly diminished function over time, mitochondria generate less energy as we age, posing real problems for older women considering pregnancy. It is possible that this declining energy could be responsible for declining fertility as women age. Energy may decline because mitochondria fail and the cell’s way of keeping them healthy become less efficient as we age. This review summarizes what is known about mitochondrial quality control in developing eggs as they age. In the future, understanding how the best mitochondria are selected and maintained in the egg, and hence the future baby, may enable older women with or without mitochondrial problems, to have healthy children.

Mitochondria are the primary sites for cellular energy production and are required for many essential cellular processes. Mitochondrial DNA (mtDNA) is a 16.6 kb circular DNA molecule that encodes only 13 gene products of the approximately 90 different proteins of the respiratory chain complexes and an estimated 1,200 mitochondrial proteins. MtDNA is, however, crucial for organismal development, normal function, and survival. MtDNA maintenance requires mitochondrially targeted nuclear DNA repair enzymes, a mtDNA replisome that is unique to mitochondria, and systems that control mitochondrial morphology and quality control. Here, we provide an overview of the current literature on mtDNA repair and transcription machineries and discuss how dynamic functional interactions between the components of these systems regulate mtDNA maintenance and transcription. A profound understanding of the molecular mechanisms that control mtDNA maintenance and transcription is important as loss of mtDNA integrity is implicated in normal process of aging, inflammation, and the etiology and pathogenesis of a number of diseases.

Abstract Mitochondrial DNA (mtDNA) replication stalling is considered an initial step in the formation of mtDNA deletions that associate with genetic inherited disorders and aging. However, the molecular details of how stalled replication forks lead to mtDNA deletions accumulation are still unclear. Mitochondrial DNA deletion breakpoints preferentially occur at sequence motifs predicted to form G-quadruplexes (G4s), four-stranded nucleic acid structures that can fold in guanine-rich regions. Whether mtDNA G4s form in vivo and their potential implication for mtDNA instability is still under debate. In here, we developed new tools to map G4s in the mtDNA of living cells. We engineered a G4-binding protein targeted to the mitochondrial matrix of a human cell line and established the mtG4-ChIP method, enabling the determination of mtDNA G4s under different cellular conditions. Our results are indicative of transient mtDNA G4 formation in human cells. We demonstrate that mtDNA-specific replication stalling increases formation of G4s, particularly in the major arc. Moreover, elevated levels of G4 block the progression of the mtDNA replication fork and cause mtDNA loss. We conclude that stalling of the mtDNA replisome enhances mtDNA G4 occurrence, and that G4s not resolved in a timely manner can have a negative impact on mtDNA integrity.

Recent evidence suggests that iron-sulfur clusters (ISCs) in DNA replicative proteins sense DNA-mediated charge transfer to modulate nuclear DNA replication. In the mitochondrial DNA replisome, only the replicative DNA helicase (mtDNA helicase) from Drosophila melanogaster (Dm) has been shown to contain an ISC in its N-terminal, primase-like domain (NTD). In this report, we confirm the presence of the ISC and demonstrate the importance of a metal cofactor in the structural stability of the Dm mtDNA helicase. Further, we show that the NTD also serves a role in membrane binding. We demonstrate that the NTD binds to asolectin liposomes, which mimic phospholipid membranes, through electrostatic interactions. Notably, membrane binding is more specific with increasing cardiolipin content, which is characteristically high in the mitochondrial inner membrane (MIM). We suggest that the N-terminal domain of the mtDNA helicase interacts with the MIM to recruit mtDNA and initiate mtDNA replication. Furthermore, Dm NUBPL, the known ISC donor for respiratory complex I and a putative donor for Dm mtDNA helicase, was identified as a peripheral membrane protein that is likely to execute membrane-mediated ISC delivery to its target proteins.