Human pathologies frequently exhibit mutations in mitochondrial DNA (mtDNA), often correlated with the aging process. Mitochondrial DNA's deletion mutations cause the loss of genes indispensable for proper mitochondrial operations. The documented database of deletion mutations surpasses 250, with the widespread deletion emerging as the most frequent mitochondrial DNA deletion implicated in disease. The deletion action entails the removal of 4977 base pairs within the mtDNA structure. Prior research has exhibited that UVA light exposure can stimulate the production of the prevalent deletion. Concurrently, imperfections in mtDNA replication and repair are contributors to the formation of the prevalent deletion. In contrast, the molecular mechanisms governing this deletion's formation are poorly characterized. The chapter's technique involves applying physiological UVA doses to human skin fibroblasts, followed by quantitative PCR to find the common deletion.
Mitochondrial DNA (mtDNA) depletion syndromes (MDS) exhibit a relationship with irregularities in the metabolism of deoxyribonucleoside triphosphate (dNTP). 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. In mouse muscle, a sensitive method for the concurrent analysis of all four dNTPs, along with all four ribonucleoside triphosphates (NTPs), is reported, using the combination of hydrophilic interaction liquid chromatography and triple quadrupole mass spectrometry. Detecting NTPs simultaneously empowers their application as internal benchmarks for the normalization of dNTP measurements. Other tissues and organisms can also utilize this methodology for determining dNTP and NTP pool levels.
For almost two decades, two-dimensional neutral/neutral agarose gel electrophoresis (2D-AGE) has been used to examine animal mitochondrial DNA's replication and maintenance, yet its full potential remains untapped. The methodology detailed here involves a series of steps, including DNA isolation, two-dimensional neutral/neutral agarose gel electrophoresis, Southern hybridization analysis, and final interpretation of results. In addition, examples showcasing the use of 2D-AGE to examine the varied facets of mitochondrial DNA maintenance and regulation are offered.
By manipulating the copy number of mitochondrial DNA (mtDNA) in cultured cells, utilizing substances that hinder DNA replication, we can effectively probe various aspects of mtDNA maintenance. Employing 2',3'-dideoxycytidine (ddC), we observed a reversible reduction in mitochondrial DNA (mtDNA) copy numbers within human primary fibroblast and HEK293 cell cultures. Upon cessation of ddC treatment, cells depleted of mitochondrial DNA (mtDNA) endeavor to restore their normal mtDNA copy count. Assessing the repopulation of mtDNA provides a valuable insight into the enzymatic function of the mtDNA replication mechanism.
The endosymbiotic origin of eukaryotic mitochondria is evident in their possession of their own genetic material, mitochondrial DNA (mtDNA), and intricate systems for maintaining and expressing this DNA. Essential subunits of the mitochondrial oxidative phosphorylation system are all encoded by mtDNA molecules, despite the limited number of proteins involved. We present protocols, here, for the monitoring of DNA and RNA synthesis in intact, isolated mitochondria. For understanding the mechanisms and regulation of mtDNA maintenance and its expression, organello synthesis protocols are valuable techniques.
The integrity of mitochondrial DNA (mtDNA) replication is critical for the effective operation of the oxidative phosphorylation system. Obstacles in mitochondrial DNA (mtDNA) maintenance, including replication interruptions triggered by DNA damage, affect its vital function and can potentially result in a range of diseases. An in vitro system recreating mtDNA replication can be used to examine the mtDNA replisome's management of, for instance, oxidative or UV-damaged DNA. We elaborate, in this chapter, a detailed protocol for exploring the bypass of diverse DNA damages via a rolling circle replication assay. Leveraging purified recombinant proteins, the assay is adjustable to examining multiple facets of mtDNA upkeep.
The unwinding of the mitochondrial genome's double helix, a task crucial for DNA replication, is performed by the helicase TWINKLE. 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. The methods described below aim to determine the TWINKLE helicase and ATPase activities. In the helicase assay, a radiolabeled oligonucleotide, annealed to a single-stranded M13mp18 DNA template, is subjected to incubation with TWINKLE. Using gel electrophoresis and autoradiography, the oligonucleotide, displaced by TWINKLE, is visualized. A colorimetric assay, designed to quantify phosphate release stemming from ATP hydrolysis by TWINKLE, is employed to gauge the ATPase activity of this enzyme.
Mirroring their evolutionary heritage, mitochondria house their own genome (mtDNA), tightly packed within the mitochondrial chromosome or nucleoid structure (mt-nucleoid). Many mitochondrial disorders are defined by the disruption of mt-nucleoids, which might stem from direct alterations in genes controlling mtDNA organization, or from the interference with other vital mitochondrial proteins. Multibiomarker approach Consequently, alterations in the mt-nucleoid's form, placement, and structure are a characteristic manifestation of numerous human diseases and can be leveraged as a criterion for cellular fitness. Through its exceptional resolution, electron microscopy allows a precise determination of the spatial and structural characteristics of all cellular elements. In recent research, ascorbate peroxidase APEX2 has been utilized to improve the contrast in transmission electron microscopy (TEM) images by triggering diaminobenzidine (DAB) precipitation. DAB's capacity for osmium accumulation during classical electron microscopy sample preparation results in strong contrast within transmission electron microscopy images, a consequence of its high electron density. A tool has been successfully developed using the fusion of mitochondrial helicase Twinkle with APEX2 to target mt-nucleoids among nucleoid proteins, allowing visualization of these subcellular structures with high-contrast and electron microscope resolution. Hydrogen peroxide (H2O2) triggers APEX2 to polymerize DAB, leading to a brown precipitate observable in particular mitochondrial matrix regions. For the production of murine cell lines expressing a transgenic variant of Twinkle, a thorough procedure is supplied. This enables targeted visualization of mt-nucleoids. Furthermore, we detail the essential procedures for validating cell lines before electron microscopy imaging, alongside illustrative examples of anticipated outcomes.
MtDNA's replication and transcription processes take place in the compact nucleoprotein complexes of mitochondrial nucleoids. Past proteomic strategies for the identification of nucleoid proteins have been explored; however, a unified list encompassing nucleoid-associated proteins has not materialized. In this description, we explore a proximity-biotinylation assay, BioID, which aids in pinpointing interacting proteins that are close to mitochondrial nucleoid proteins. A protein of interest, augmented with a promiscuous biotin ligase, creates a covalent bond between biotin and lysine residues of adjacent proteins. By employing a biotin-affinity purification technique, biotinylated proteins can be further enriched and their identity confirmed via mass spectrometry. Transient and weak interactions can be identified by BioID, which is also capable of detecting alterations in these interactions under various cellular treatments, protein isoform variations, or pathogenic mutations.
Mitochondrial transcription factor A (TFAM), a protein that binds mitochondrial DNA (mtDNA), undertakes a dual function, initiating mitochondrial transcription and upholding mtDNA stability. TFAM's direct connection to mtDNA facilitates the acquisition of useful knowledge regarding its DNA-binding capabilities. Employing recombinant TFAM proteins, this chapter details two in vitro assay methodologies: an electrophoretic mobility shift assay (EMSA) and a DNA-unwinding assay. Both techniques hinge on the use of simple agarose gel electrophoresis. Investigations into the effects of mutations, truncations, and post-translational modifications on this vital mtDNA regulatory protein are conducted using these tools.
In the organization and compaction of the mitochondrial genome, mitochondrial transcription factor A (TFAM) holds a primary role. airway infection Nonetheless, only a limited number of uncomplicated and easily accessible methods are available to quantify and observe TFAM-driven DNA condensation. Straightforward in its implementation, Acoustic Force Spectroscopy (AFS) is a single-molecule force spectroscopy technique. It's possible to track and quantify the mechanical properties of numerous individual protein-DNA complexes in a parallel fashion. High-throughput single-molecule Total Internal Reflection Fluorescence (TIRF) microscopy allows for a real-time view of TFAM's movements on DNA, a feat impossible with traditional biochemical tools. Irinotecan This document provides a comprehensive description of the establishment, execution, and analysis of AFS and TIRF measurements, specifically focusing on DNA compaction regulated by TFAM.
Mitochondria's unique genetic material, mtDNA, is tightly organized within cellular structures called nucleoids. Fluorescence microscopy allows for in situ visualization of nucleoids, yet super-resolution microscopy, particularly stimulated emission depletion (STED), has ushered in an era of sub-diffraction resolution visualization for these nucleoids.