In several human health conditions, mitochondrial DNA (mtDNA) mutations are identified, and their presence is associated with the aging process. Genetic deletions within mitochondrial DNA diminish the availability of necessary genes critical for mitochondrial function. A substantial number of deletion mutations—exceeding 250—have been found, and the common deletion is the most frequent mtDNA deletion known to cause diseases. The removal of 4977 mtDNA base pairs is accomplished by this deletion. Past studies have revealed a correlation between UVA radiation exposure and the development of the typical deletion. Subsequently, inconsistencies in mitochondrial DNA replication and repair procedures are connected to the production of the prevalent deletion. Nevertheless, the molecular processes responsible for this deletion are not well-defined. This chapter describes the procedure of exposing human skin fibroblasts to physiological doses of UVA, subsequently analyzing for the common deletion using quantitative PCR.

Problems in the deoxyribonucleoside triphosphate (dNTP) metabolic process are frequently observed in cases 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. Therefore, the levels of dNTPs in the tissues of healthy and MDS-affected animals are essential for investigating the processes of mtDNA replication, studying disease advancement, and creating therapeutic interventions. In this work, a sensitive method is detailed for simultaneously determining all four dNTPs and all four ribonucleoside triphosphates (NTPs) in mouse muscles, leveraging hydrophilic interaction liquid chromatography and triple quadrupole mass spectrometry. Coincidental NTP detection facilitates their use as internal benchmarks for adjusting dNTP levels. For the determination of dNTP and NTP pools, this method is applicable to diverse tissues and organisms.

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. This method involves a sequence of steps, starting with DNA extraction, advancing through two-dimensional neutral/neutral agarose gel electrophoresis, and concluding with Southern blot analysis and interpretation of the results. We also furnish examples demonstrating the practicality of 2D-AGE in investigating the distinct features of mtDNA preservation and governance.

A valuable approach to studying mtDNA maintenance involves manipulating the copy number of mitochondrial DNA (mtDNA) in cultured cells via the application of substances that interfere with DNA replication. We investigate the effect of 2',3'-dideoxycytidine (ddC) on mtDNA copy number, demonstrating a reversible decrease in human primary fibroblasts and HEK293 cells. Stopping the use of ddC triggers an attempt by cells lacking sufficient mtDNA to return to their usual mtDNA copy numbers. Assessing the repopulation of mtDNA provides a valuable insight into the enzymatic function of the mtDNA replication mechanism.

Endosymbiotic in origin, eukaryotic mitochondria possess their own genetic code, mitochondrial DNA, and mechanisms dedicated to the DNA's maintenance and expression. Mitochondrial DNA molecules encode a restricted set of proteins, all of which are indispensable components of the mitochondrial oxidative phosphorylation system. This report outlines protocols for observing DNA and RNA synthesis processes in intact, isolated mitochondria. The study of mtDNA maintenance and expression mechanisms and regulation finds valuable tools in organello synthesis protocols.

Accurate mitochondrial DNA (mtDNA) replication is indispensable for the correct functioning 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. Employing a laboratory-based, reconstituted mtDNA replication system, researchers can examine how the mtDNA replisome navigates issues like oxidative or ultraviolet DNA damage. This chapter's detailed protocol outlines how to investigate the bypass of different DNA damage types through the use of a rolling circle replication assay. Purified recombinant proteins empower the assay, which can be tailored for investigating various facets of mtDNA maintenance.

During the process of mitochondrial DNA replication, the crucial helicase TWINKLE separates the double-stranded DNA. 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. Our approach to investigating TWINKLE's helicase and ATPase functions is outlined here. For the helicase assay procedure, a single-stranded DNA template from M13mp18, having a radiolabeled oligonucleotide annealed to it, is combined with TWINKLE, then incubated. The oligonucleotide, subsequently visualized via gel electrophoresis and autoradiography, will be displaced by TWINKLE. By quantifying the phosphate released during the hydrolysis of ATP by TWINKLE, a colorimetric assay provides a means of measuring the ATPase activity of TWINKLE.

Reflecting their evolutionary ancestry, mitochondria retain their own genetic material (mtDNA), concentrated within the mitochondrial chromosome or the 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. Infection and disease risk assessment Therefore, fluctuations in the mt-nucleoid's morphology, arrangement, and composition are prevalent in numerous human diseases and can be utilized to gauge cellular health. Electron microscopy, in achieving the highest possible resolution, allows for the determination of the spatial and structural characteristics of all cellular components. Transmission electron microscopy (TEM) contrast has been improved in recent studies through the application of ascorbate peroxidase APEX2, which catalyzes diaminobenzidine (DAB) precipitation. Osmium accumulation in DAB, a characteristic of classical electron microscopy sample preparation, yields significant contrast enhancement in transmission electron microscopy, owing to the substance's high electron density. Among the nucleoid proteins, the successfully targeted mt-nucleoids by a fusion protein comprising APEX2 and the mitochondrial helicase Twinkle allows high-contrast visualization of these subcellular structures using electron microscope resolution. The presence of H2O2 facilitates APEX2-catalyzed DAB polymerization, yielding a brown precipitate, which is easily visualized in specific mitochondrial matrix locations. 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. We additionally outline the complete set of procedures for validating cell lines prior to electron microscopy imaging, complete with examples demonstrating the anticipated outcomes.

The compact nucleoprotein complexes that constitute mitochondrial nucleoids contain, replicate, and transcribe mtDNA. Past proteomic strategies for the identification of nucleoid proteins have been explored; however, a unified list encompassing nucleoid-associated proteins has not materialized. This proximity-biotinylation assay, BioID, is described here, facilitating the identification of nearby proteins associated with mitochondrial nucleoid proteins. By fusing a promiscuous biotin ligase to a protein of interest, biotin is covalently added to lysine residues of its neighboring proteins. Utilizing biotin-affinity purification, biotinylated proteins can be further enriched and identified by means of mass spectrometry. The identification of transient and weak interactions, a function of BioID, further permits the examination of modifications to these interactions under disparate cellular manipulations, protein isoform variations or in the context of pathogenic variants.

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. Because of TFAM's direct connection to mtDNA, examining its DNA-binding capabilities provides useful data. 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. This key mtDNA regulatory protein is scrutinized for its reactivity to mutations, truncations, and post-translational modifications using these methods.

Mitochondrial transcription factor A (TFAM) actively participates in the arrangement and compression of the mitochondrial genetic material. Pomalidomide Even so, a limited number of uncomplicated and widely usable methods exist to observe and determine the degree of DNA compaction regulated by TFAM. AFS, a straightforward method, is a single-molecule force spectroscopy technique. Simultaneous monitoring of numerous individual protein-DNA complexes permits the assessment of their mechanical properties. TFAM's movements on DNA can be observed in real-time through high-throughput, single-molecule TIRF microscopy, a technique inaccessible to traditional biochemical approaches. Normalized phylogenetic profiling (NPP) This report provides a detailed explanation for establishing, conducting, and evaluating AFS and TIRF measurements to explore the impact of TFAM on DNA compaction.

Equipped with their own DNA, mitochondrial DNA or mtDNA, this genetic material is organized in nucleoid formations. Fluorescence microscopy enables the in situ visualization of nucleoids, but the development and application of stimulated emission depletion (STED) super-resolution microscopy has made possible the visualization of nucleoids at the sub-diffraction resolution level.