The aging process is related to mitochondrial DNA (mtDNA) mutations, which are frequently observed in various human health problems. Mitochondrial DNA deletion mutations are responsible for the removal of essential genes, consequently affecting mitochondrial function. 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. Studies conducted in the past have indicated that exposure to UVA light can lead to the creation of the frequent deletion. Similarly, irregularities in the mechanisms of mtDNA replication and repair are directly involved in the emergence of the common deletion. While this deletion's formation occurs, the associated molecular mechanisms are poorly understood. This chapter presents a method of irradiating human skin fibroblasts with physiological UVA levels, and using quantitative PCR to detect the associated frequent deletion.

Deoxyribonucleoside triphosphate (dNTP) metabolic flaws are linked to a variety of mitochondrial DNA (mtDNA) depletion syndromes (MDS). The muscles, liver, and brain are targets of these disorders, and the dNTP concentrations within these tissues are naturally low, consequently making accurate measurement difficult. In sum, data about dNTP concentrations in the tissues of both healthy and MDS-affected animals are critical for examining the mechanisms of mtDNA replication, assessing the progression of the disease, and creating 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. Simultaneous NTP detection allows for their utilization as internal standards to normalize the amounts of dNTPs. Measuring dNTP and NTP pools in other tissues and organisms is facilitated by this applicable method.

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. Our description of this method covers each stage, from DNA isolation to two-dimensional neutral/neutral agarose gel electrophoresis, Southern hybridization, and finally, the analysis of the derived data. We also furnish examples demonstrating the practicality of 2D-AGE in investigating the distinct features of mtDNA preservation and governance.

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. This report elucidates the utilization of 2',3'-dideoxycytidine (ddC) to effect a reversible decline in mtDNA copy number in both human primary fibroblasts and HEK293 cells. Terminating the application of ddC stimulates the mtDNA-depleted cells to recover their usual mtDNA copy levels. The process of mtDNA repopulation dynamically reflects the enzymatic efficiency of the mtDNA replication system.

Eukaryotic mitochondria, of endosymbiotic ancestry, encompass their own genetic material, namely mitochondrial DNA, and possess specialized systems for the upkeep and translation of this genetic material. MtDNA's limited protein repertoire is nonetheless crucial, with all encoded proteins being essential components of the mitochondrial oxidative phosphorylation system. This report outlines protocols for observing DNA and RNA synthesis processes in intact, isolated mitochondria. In the exploration of mtDNA maintenance and expression, organello synthesis protocols prove to be significant tools in deciphering mechanisms and regulation.

The integrity of mitochondrial DNA (mtDNA) replication is critical for the effective operation of the oxidative phosphorylation system. Failures in mtDNA maintenance, particularly replication disruptions stemming from DNA damage, impede its essential role and could potentially result in disease conditions. Researchers can investigate the mtDNA replisome's handling of oxidative or UV-damaged DNA using a recreated mtDNA replication system outside of a living cell. The methodology for studying DNA damage bypass, employing a rolling circle replication assay, is meticulously detailed in this chapter. Purified recombinant proteins empower the assay, which can be tailored for investigating various facets of mtDNA maintenance.

DNA replication of the mitochondrial genome hinges on the essential helicase TWINKLE, which unwinds its double-stranded structure. To gain mechanistic understanding of TWINKLE's function at the replication fork, in vitro assays using purified recombinant forms of the protein have proved invaluable. The following methods are presented for probing the helicase and ATPase activities of the TWINKLE enzyme. During the helicase assay, TWINKLE is incubated alongside a radiolabeled oligonucleotide, which is previously annealed to an M13mp18 single-stranded DNA template. TWINKLE displaces the oligonucleotide, and this displacement is subsequently visualized by employing gel electrophoresis and autoradiography. A colorimetric method serves to measure the ATPase activity of TWINKLE, by quantifying the phosphate that is released during TWINKLE's ATP hydrolysis.

Bearing a resemblance to their evolutionary origins, mitochondria possess their own genetic material (mtDNA), condensed into the mitochondrial chromosome or nucleoid (mt-nucleoid). Mutations directly impacting mtDNA organizational genes or interference with critical mitochondrial proteins contribute to the disruption of mt-nucleoids observed in numerous mitochondrial disorders. Selleck GS-9674 Hence, modifications to the mt-nucleoid's shape, placement, and design are commonplace in diverse human diseases, and this can serve as a sign of the cell's viability. Electron microscopy is instrumental in reaching the highest resolution possible, providing information on the spatial structure of every cellular component. Recent research has explored the use of ascorbate peroxidase APEX2 to enhance transmission electron microscopy (TEM) contrast by catalyzing the precipitation of diaminobenzidine (DAB). 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. Twinkle, a mitochondrial helicase, fused with APEX2, has effectively targeted mt-nucleoids among the nucleoid proteins, offering a tool for high-contrast visualization of these subcellular structures at electron microscope resolution. In the mitochondria, a brown precipitate forms due to APEX2-catalyzed DAB polymerization in the presence of hydrogen peroxide, localizable in specific regions of the matrix. This document provides a detailed protocol for generating murine cell lines expressing a modified Twinkle protein, allowing for the visualization and targeting of mitochondrial nucleoids. We also furnish a detailed account of the indispensable procedures for validating cell lines before embarking on electron microscopy imaging, including examples of anticipated outcomes.

Mitochondrial nucleoids, the site of mtDNA replication and transcription, are dense nucleoprotein complexes. Prior proteomic investigations into nucleoid proteins have been numerous; nonetheless, a comprehensive catalog of nucleoid-associated proteins has yet to be established. Through a proximity-biotinylation assay, BioID, we describe the method for identifying proteins interacting closely with mitochondrial nucleoid proteins. 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. Utilizing biotin-affinity purification, biotinylated proteins can be further enriched and identified by means of 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.

TFAM, a protein that binds to mitochondrial DNA (mtDNA), is crucial for both initiating mitochondrial transcription and preserving mtDNA integrity. Since TFAM has a direct interaction with mtDNA, evaluating its DNA-binding capacity offers valuable insights. The chapter describes two in vitro assay procedures, an electrophoretic mobility shift assay (EMSA) and a DNA-unwinding assay, using recombinant TFAM proteins. Both methods require the standard technique of agarose gel electrophoresis. The use of these approaches allows for an exploration of the effects of mutations, truncations, and post-translational modifications on this critical mtDNA regulatory protein.

Mitochondrial transcription factor A (TFAM) orchestrates the arrangement and compactness of the mitochondrial genome. medicine information services Yet, a restricted number of simple and accessible techniques are available for quantifying and observing the DNA compaction that TFAM is responsible for. Acoustic Force Spectroscopy (AFS), a method for single-molecule force spectroscopy, possesses a straightforward nature. One can monitor a multitude of individual protein-DNA complexes simultaneously, enabling the quantification of their mechanical characteristics. Real-time visualization of TFAM's interactions with DNA, made possible by high-throughput single-molecule TIRF microscopy, is unavailable with classical biochemical tools. E coli infections We elaborate on the setup, procedure, and analysis of AFS and TIRF measurements for elucidating how TFAM affects the compaction of DNA.

The DNA within mitochondria, specifically mtDNA, is compactly packaged inside structures known as nucleoids. Even though fluorescence microscopy allows for in situ observations of nucleoids, the incorporation of super-resolution microscopy, specifically stimulated emission depletion (STED), has unlocked a new potential for imaging nucleoids with a sub-diffraction resolution.