In several human health conditions, mitochondrial DNA (mtDNA) mutations are identified, and their presence is associated with the aging process. Mutations deleting portions of mitochondrial DNA result in the absence of necessary genes for mitochondrial processes. The reported deletion mutations exceed 250, with the prevailing deletion mutation being the most frequent mtDNA deletion associated with disease. This deletion operation removes a section of mtDNA, specifically 4977 base pairs. UVA radiation has been previously shown to encourage the formation of the frequently occurring deletion. Additionally, deviations in mtDNA replication and repair mechanisms contribute to the formation of the common deletion. Despite this, the molecular mechanisms driving the formation of this deletion are inadequately characterized. Human skin fibroblasts are irradiated with physiological UVA doses in this chapter, and the resulting common deletion is detected using quantitative PCR.
The presence of mitochondrial DNA (mtDNA) depletion syndromes (MDS) is sometimes accompanied by impairments in deoxyribonucleoside triphosphate (dNTP) metabolic functions. These disorders cause issues for the muscles, liver, and brain, and dNTP concentrations in these tissues are already, naturally, low, which makes measurement difficult. Consequently, knowledge of dNTP concentrations within the tissues of both healthy and MDS-affected animals is crucial for understanding the mechanics of mtDNA replication, tracking disease progression, and creating effective therapeutic strategies. We introduce a delicate methodology for simultaneously assessing all four deoxynucleoside triphosphates (dNTPs) and the four ribonucleoside triphosphates (NTPs) within mouse muscle tissue, employing hydrophilic interaction liquid chromatography coupled with a triple quadrupole mass spectrometer. Concurrent NTP detection provides them with the capacity to act as internal standards for the normalization of dNTP levels. This method allows for the assessment of dNTP and NTP pools in other tissues and a wide range of organisms.
For nearly two decades, two-dimensional neutral/neutral agarose gel electrophoresis (2D-AGE) has been employed to analyze the processes of animal mitochondrial DNA replication and maintenance, with its full potential yet to be fully exploited. From the initial DNA isolation process to the subsequent two-dimensional neutral/neutral agarose gel electrophoresis, the subsequent Southern blot hybridization, and the conclusive data analysis, we detail the procedure. We additionally present instances of 2D-AGE's application in examining the diverse characteristics of mtDNA maintenance and regulation.
To understand diverse facets of mtDNA maintenance, manipulation of mitochondrial DNA (mtDNA) copy number in cultured cells using substances that interrupt DNA replication proves to be a valuable tool. The present work examines how 2',3'-dideoxycytidine (ddC) can induce a reversible decrement in mitochondrial DNA (mtDNA) content in human primary fibroblasts and human embryonic kidney (HEK293) cells. Following the discontinuation of ddC administration, cells exhibiting mtDNA depletion seek to regain their standard mtDNA copy numbers. The dynamics of mtDNA repopulation offers a significant measure for evaluating the enzymatic effectiveness of the mtDNA replication machinery.
Mitochondria, eukaryotic cell components with endosymbiotic origins, contain their own genetic material, mtDNA, and systems specialized in its upkeep and genetic expression. The proteins encoded by mtDNA molecules are, while few in number, all critical parts of the mitochondrial oxidative phosphorylation machinery. Intact, isolated mitochondria are the subject of the protocols described here for monitoring DNA and RNA synthesis. The study of mtDNA maintenance and expression mechanisms and regulation finds valuable tools in organello synthesis protocols.
The cellular process of mitochondrial DNA (mtDNA) replication must be accurate for the oxidative phosphorylation system to function correctly. Challenges related to mtDNA upkeep, including replication stagnation upon encountering DNA damage, impair its crucial role, which can potentially initiate disease processes. Employing a laboratory-based, reconstituted mtDNA replication system, researchers can examine how the mtDNA replisome navigates issues like oxidative or ultraviolet DNA damage. We elaborate, in this chapter, a detailed protocol for exploring the bypass of diverse DNA damages via a rolling circle replication assay. The assay's capability rests on purified recombinant proteins and it can be adjusted to the investigation of different aspects of mtDNA maintenance.
Essential for the replication of mitochondrial DNA, TWINKLE helicase is responsible for disentangling the duplex genome. Purified recombinant forms of the protein have served as instrumental components in in vitro assays that have provided mechanistic insights into TWINKLE's function at the replication fork. This paper demonstrates methods for characterizing the helicase and ATPase properties of TWINKLE. A radiolabeled oligonucleotide, annealed to an M13mp18 single-stranded DNA template, is incubated with TWINKLE for the helicase assay. The oligonucleotide, subsequently visualized via gel electrophoresis and autoradiography, will be displaced by TWINKLE. A colorimetric assay for the quantification of phosphate released during ATP hydrolysis by TWINKLE, is employed to determine its ATPase activity.
Recalling their evolutionary roots, mitochondria carry their own genetic code (mtDNA), condensed into the mitochondrial chromosome or the nucleoid (mt-nucleoid). A hallmark of many mitochondrial disorders is the disruption of mt-nucleoids, which can arise from direct mutations in genes responsible for mtDNA structure or from interference with other essential mitochondrial proteins. Oil remediation Thusly, changes in the mt-nucleoid's morphology, dissemination, and composition are frequently present in various human maladies, and they can be exploited to assess cellular proficiency. Electron microscopy is instrumental in reaching the highest resolution possible, providing information on the spatial structure of every cellular component. Transmission electron microscopy (TEM) contrast has been improved in recent studies through the application of ascorbate peroxidase APEX2, which catalyzes diaminobenzidine (DAB) precipitation. During the classical electron microscopy sample preparation process, DAB's accumulation of osmium elevates its electron density, ultimately producing a strong contrast effect in transmission electron microscopy. To visualize mt-nucleoids with high contrast and electron microscope resolution, a tool utilizing the fusion of mitochondrial helicase Twinkle with APEX2 has been successfully implemented among nucleoid proteins. When hydrogen peroxide is present, APEX2 catalyzes the polymerization of DAB, forming a brown precipitate that can be visualized within specific areas of the mitochondrial matrix. A comprehensive protocol is outlined for the creation of murine cell lines expressing a transgenic Twinkle variant, facilitating the visualization and targeting of mt-nucleoids. We also comprehensively detail each step needed for validating cell lines before electron microscopy imaging, and provide examples of the anticipated outcomes.
MtDNA's replication and transcription processes take place in the compact nucleoprotein complexes of mitochondrial nucleoids. Previous proteomic endeavors to identify nucleoid proteins have been conducted; however, a standardized list of nucleoid-associated proteins is still lacking. We explain a proximity-biotinylation assay, BioID, to identify proteins that are in close proximity to mitochondrial nucleoid proteins. A protein of interest, to which a promiscuous biotin ligase is attached, forms a covalent link between biotin and lysine residues of its immediately adjacent proteins. Biotinylated proteins are further enriched by a biotin-affinity purification protocol and subsequently identified through mass spectrometry. BioID's application in detecting transient and weak interactions extends to analyzing changes in these interactions resulting from various cellular treatments, different protein isoforms, or the presence of pathogenic variants.
Mitochondrial transcription factor A (TFAM), a protein that binds mitochondrial DNA, is instrumental in the initiation of mitochondrial transcription and in safeguarding mtDNA's integrity. Considering TFAM's direct interaction with mitochondrial DNA, understanding its DNA-binding capacity proves helpful. Two assay methodologies, an electrophoretic mobility shift assay (EMSA) and a DNA-unwinding assay, are explored in this chapter, both utilizing recombinant TFAM proteins. Each requires a basic agarose gel electrophoresis procedure. The effects of mutations, truncation, and post-translational modifications on the function of this essential mtDNA regulatory protein are explored using these instruments.
The mitochondrial genome's structure and packing depend heavily on the action of mitochondrial transcription factor A (TFAM). genetic recombination Despite this, only a few simple and easily obtainable procedures are present for examining and evaluating the TFAM-influenced compaction of DNA. Acoustic Force Spectroscopy (AFS), a straightforward method, facilitates single-molecule force spectroscopy. Simultaneous monitoring of numerous individual protein-DNA complexes permits the assessment of their mechanical properties. Utilizing Total Internal Reflection Fluorescence (TIRF) microscopy, a high-throughput single-molecule approach, real-time observation of TFAM's movements on DNA is permitted, a significant advancement over classical biochemical tools. LY3295668 A detailed account of the setup, execution, and analysis of AFS and TIRF experiments is offered here, to investigate TFAM's role in altering DNA compaction.
Within mitochondria, the genetic material, mtDNA, is contained within specialized compartments called nucleoids. Fluorescence microscopy can visualize nucleoids in situ, but super-resolution microscopy, particularly stimulated emission depletion (STED) technology, has recently yielded the capability to observe nucleoids at a resolution exceeding the diffraction limit.