Mutations in mitochondrial DNA (mtDNA) are prevalent in various human ailments and are linked to the aging process. Essential genes for mitochondrial function are absent due to deletion mutations within the mitochondrial DNA. Reports indicate over 250 deletion mutations, the most frequent of which is the common mtDNA deletion implicated in disease. This deletion operation removes a section of mtDNA, specifically 4977 base pairs. Earlier research has confirmed that UVA radiation can promote the occurrence of the widespread deletion. Furthermore, discrepancies in mitochondrial DNA replication and repair procedures are implicated in the development of the widespread deletion. Although this deletion forms, the molecular mechanisms involved in its formation are inadequately described. The chapter outlines a procedure for exposing human skin fibroblasts to physiological UVA doses, culminating in the quantitative PCR detection of the frequent deletion.

Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are characterized by defects 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. Subsequently, the quantities of dNTPs within the tissues of healthy and MDS-affected animals provide crucial insights into the processes of mtDNA replication, the study of disease progression, and the creation of therapeutic applications. Using hydrophilic interaction liquid chromatography coupled with triple quadrupole mass spectrometry, a sensitive method for the simultaneous determination of all four dNTPs and all four ribonucleoside triphosphates (NTPs) in mouse muscle is presented. The simultaneous observation of NTPs allows them to function as internal controls for the standardization of dNTP quantities. This method's application encompasses the measurement of dNTP and NTP pools in various organisms and tissues.

Nearly two decades of application in the analysis of animal mitochondrial DNA replication and maintenance processes have been observed with two-dimensional neutral/neutral agarose gel electrophoresis (2D-AGE), yet its full potential has not been fully utilized. 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. We also provide examples that illustrate the utility of 2D-AGE in examining the different characteristics of mitochondrial DNA preservation and regulation.

Substances that impede DNA replication can be used to modulate mtDNA copy number in cultured cells, making this a useful tool to study mtDNA maintenance processes. We explore the use of 2',3'-dideoxycytidine (ddC) for achieving a reversible reduction in mitochondrial DNA (mtDNA) levels in human primary fibroblast and HEK293 cell lines. After the cessation of ddC therapy, cells lacking normal mtDNA quantities attempt to reestablish normal mtDNA copy levels. MtDNA repopulation patterns yield a valuable measurement of the enzymatic capabilities of the mtDNA replication machinery.

Eukaryotic organelles, mitochondria, are products of endosymbiosis, containing their own genetic material (mtDNA) and systems specifically for mtDNA's upkeep and translation. MtDNA molecules' encoded proteins, though limited in quantity, are all fundamental to the mitochondrial oxidative phosphorylation system's operation. Protocols for observing DNA and RNA synthesis within intact, isolated mitochondria are detailed below. Research into mtDNA maintenance and expression mechanisms and their regulation benefits significantly from the use of organello synthesis protocols.

For the oxidative phosphorylation system to operate optimally, faithful mitochondrial DNA (mtDNA) replication is paramount. Issues with the preservation of mitochondrial DNA (mtDNA), like replication blocks due to DNA damage, compromise its essential function and can potentially lead to diseases. A laboratory-generated mtDNA replication system provides a means of studying the mtDNA replisome's response to oxidative or UV-induced DNA lesions. This chapter details a comprehensive protocol for studying the bypass of various DNA lesions using a rolling circle replication assay. Purified recombinant proteins form the basis of this assay, which is adaptable to studying diverse facets of mtDNA maintenance.

Essential for the replication of mitochondrial DNA, TWINKLE helicase is responsible for disentangling the duplex genome. For gaining mechanistic insights into the role of TWINKLE at the replication fork, in vitro assays using purified recombinant proteins have been essential tools. We detail methods for investigating the helicase and ATPase functions of TWINKLE. The helicase assay protocol entails the incubation of TWINKLE with a radiolabeled oligonucleotide that is hybridized to a single-stranded M13mp18 DNA template. TWINKLE displaces the oligonucleotide, and this displacement is subsequently visualized by employing gel electrophoresis and autoradiography. A colorimetric assay for the quantification of phosphate released during ATP hydrolysis by TWINKLE, is employed to determine its ATPase activity.

In echoing their evolutionary roots, mitochondria are equipped with their own genome (mtDNA), compacted within the mitochondrial chromosome or the nucleoid (mt-nucleoid). Mitochondrial disorders frequently involve disruptions of mt-nucleoids, arising from direct mutations within genes essential for mtDNA structure or interference with other indispensable proteins for mitochondrial processes. transhepatic artery embolization Subsequently, variations in the mt-nucleoid's morphology, dispersion, and construction are frequently encountered in numerous human diseases, and this can be used as an indicator of cellular function. Electron microscopy is instrumental in reaching the highest resolution possible, providing information on the spatial structure of every cellular component. To boost transmission electron microscopy (TEM) contrast, ascorbate peroxidase APEX2 has recently been used to facilitate diaminobenzidine (DAB) precipitation. In classical electron microscopy sample preparation, DAB's capacity for osmium accumulation creates a high electron density, which is essential for generating strong contrast in transmission electron microscopy. Among nucleoid proteins, the fusion of mitochondrial helicase Twinkle and APEX2 has proven successful in targeting mt-nucleoids, creating a tool that provides high-contrast visualization of these subcellular structures with electron microscope resolution. DAB polymerization, catalyzed by APEX2 in the presence of hydrogen peroxide, produces a brown precipitate which is detectable within particular regions of the mitochondrial matrix. This protocol meticulously details the generation of murine cell lines expressing a transgenic Twinkle variant, designed for the targeting and visualization of mt-nucleoids. Furthermore, we detail the essential procedures for validating cell lines before electron microscopy imaging, alongside illustrative examples of anticipated outcomes.

Within mitochondrial nucleoids, the compact nucleoprotein complexes are the sites for the replication and transcription of mtDNA. Prior studies employing proteomic techniques to identify nucleoid proteins have been carried out; nevertheless, a unified inventory of nucleoid-associated proteins has not been created. To identify interaction partners of mitochondrial nucleoid proteins, we present the proximity-biotinylation assay, BioID. The protein of interest, which is fused to a promiscuous biotin ligase, causes a covalent attachment of biotin to lysine residues of its proximal neighbors. A biotin-affinity purification step allows for the enrichment of biotinylated proteins, which can subsequently be identified by mass spectrometry. Changes in transient and weak protein interactions, as identified by BioID, can be investigated under diverse cellular treatments, protein isoforms, or pathogenic variant contexts.

A protein known as mitochondrial transcription factor A (TFAM), which binds to mtDNA, orchestrates both the initiation of mitochondrial transcription and the maintenance of mtDNA. Due to TFAM's direct engagement with mitochondrial DNA, determining its DNA-binding aptitude is informative. Two in vitro assay methods, the electrophoretic mobility shift assay (EMSA) and the DNA-unwinding assay, are explained in this chapter, employing recombinant TFAM proteins. Both methods share the common requirement 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. Oncology center However, a small selection of straightforward and readily usable methods remain for the assessment and observation of TFAM-dependent DNA compaction. Acoustic Force Spectroscopy (AFS), a method for single-molecule force spectroscopy, possesses a straightforward nature. This process allows for parallel analysis of numerous individual protein-DNA complexes, quantifying 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. MRTX1133 Ras inhibitor 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.

The mitochondria harbor their own DNA, designated mtDNA, which is compactly arranged in specialized compartments known as 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.