In several human health conditions, mitochondrial DNA (mtDNA) mutations are identified, and their presence is associated with the aging process. Deletion mutations in mtDNA sequences cause the elimination of essential genes needed for mitochondrial activities. The reported deletion mutations exceed 250, with the prevailing deletion mutation being the most frequent mtDNA deletion associated with disease. Due to this deletion, 4977 mtDNA base pairs are eradicated. 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. Nonetheless, the molecular mechanisms underlying this deletion's formation remain poorly understood. Quantitative PCR analysis is used in this chapter to detect the common deletion following UVA irradiation of physiological doses to human skin fibroblasts.

A correlation has been observed between mitochondrial DNA (mtDNA) depletion syndromes (MDS) and disruptions in the process of deoxyribonucleoside triphosphate (dNTP) metabolism. These disorders impact the muscles, liver, and brain, with dNTP concentrations already low within these tissues, presenting difficulties in measurement. 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. Employing hydrophilic interaction liquid chromatography coupled with triple quadrupole mass spectrometry, this work presents a sensitive method to evaluate all four dNTPs and all four ribonucleoside triphosphates (NTPs) in mouse muscle specimens. Coincidental NTP detection facilitates their use as internal benchmarks for adjusting dNTP levels. The application of this method extends to quantifying dNTP and NTP pools in various tissues and biological 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. 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. Examples of the application of 2D-AGE in the investigation of mtDNA's diverse maintenance and regulatory attributes are also included in our work.

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 detail the application of 2',3'-dideoxycytidine (ddC) to cause a reversible decrease in mitochondrial DNA (mtDNA) abundance in human primary fibroblasts and human embryonic kidney (HEK293) cells. Stopping the use of ddC triggers an attempt by cells lacking sufficient mtDNA to return to their usual mtDNA copy numbers. MtDNA repopulation patterns yield a valuable measurement of the enzymatic capabilities of the mtDNA replication machinery.

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 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. In the exploration of mtDNA maintenance and expression, organello synthesis protocols prove to be significant tools in deciphering mechanisms and regulation.

The accurate duplication of mitochondrial DNA (mtDNA) is fundamental to the proper operation of the cellular oxidative phosphorylation system. Difficulties in mitochondrial DNA (mtDNA) maintenance, including replication impediments caused by DNA damage, hinder its crucial role and can potentially result in disease manifestation. A reconstituted mitochondrial DNA (mtDNA) replication system in a laboratory setting allows investigation of how the mtDNA replisome handles oxidative or UV-induced DNA damage. We elaborate, in this chapter, a detailed protocol for exploring the bypass of diverse DNA damages via a rolling circle replication assay. For the assay, purified recombinant proteins provide the foundation, and it can be adjusted to analyze multiple facets of mtDNA preservation.

DNA replication of the mitochondrial genome hinges on the essential helicase TWINKLE, which unwinds its double-stranded structure. Recombinant protein forms, when used in in vitro assays, have provided crucial insights into the mechanistic workings of TWINKLE and its role at the replication fork. Techniques for exploring the helicase and ATPase functions of the TWINKLE protein are presented in this document. To conduct the helicase assay, a single-stranded M13mp18 DNA template, annealed to a radiolabeled oligonucleotide, is incubated with the enzyme TWINKLE. TWINKLE displaces the oligonucleotide, and this displacement is subsequently visualized by employing gel electrophoresis and autoradiography. To assess TWINKLE's ATPase activity, a colorimetric assay is utilized, which meticulously measures the phosphate liberated during the hydrolysis of ATP by TWINKLE.

Reflecting their evolutionary ancestry, mitochondria retain their own genetic material (mtDNA), concentrated within the mitochondrial chromosome or the nucleoid (mt-nucleoid). Disruptions in mt-nucleoids are characteristic of many mitochondrial disorders, originating either from direct alterations in the genes governing mtDNA organization or from interference with essential mitochondrial proteins. Selleck NHWD-870 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. All cellular structures' spatial and structural properties are elucidated through electron microscopy's unique ability to achieve the highest possible resolution. In recent research, ascorbate peroxidase APEX2 has been utilized to improve the contrast in transmission electron microscopy (TEM) images by triggering diaminobenzidine (DAB) precipitation. During classical electron microscopy sample preparation, DAB exhibits the capacity to accumulate osmium, resulting in strong contrast for transmission electron microscopy due to 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. The presence of H2O2 facilitates APEX2-catalyzed DAB polymerization, yielding a brown precipitate, which is easily visualized in specific mitochondrial matrix locations. We present a detailed method for generating murine cell lines carrying a transgenic Twinkle variant, specifically designed to target and visualize mt-nucleoids. In addition, we delineate every crucial step in validating cell lines before electron microscopy imaging, along with examples of expected results.

Mitochondrial nucleoids, composed of nucleoprotein complexes, are the sites for the replication, transcription, and containment of mtDNA. Previous proteomic investigations targeting nucleoid proteins have been performed; however, there is still no agreed-upon list of nucleoid-associated proteins. To identify interaction partners of mitochondrial nucleoid proteins, we present the proximity-biotinylation assay, BioID. A fused protein of interest, equipped with a promiscuous biotin ligase, chemically links biotin to the lysine residues of its nearest neighboring proteins. A biotin-affinity purification step allows for the enrichment of biotinylated proteins, which can subsequently be identified by mass spectrometry. BioID's capacity to detect transient and weak interactions extends to discerning changes in these interactions brought about by diverse cellular treatments, protein isoforms, or pathogenic variants.

Mitochondrial transcription factor A (TFAM), a protein that binds mitochondrial DNA (mtDNA), undertakes a dual function, initiating mitochondrial transcription and upholding mtDNA stability. 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 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) actively participates in the arrangement and compression of the mitochondrial genetic material. prognostic biomarker Even so, a limited number of uncomplicated and widely usable methods exist to observe and determine the degree of DNA compaction regulated by TFAM. Within the domain of single-molecule force spectroscopy, Acoustic Force Spectroscopy (AFS) is a straightforward technique. It enables the simultaneous assessment of numerous individual protein-DNA complexes and the determination of their mechanical properties. Real-time visualization of TFAM's interactions with DNA, made possible by high-throughput single-molecule TIRF microscopy, is unavailable with classical biochemical tools. skin biophysical parameters In this detailed account, we delineate the procedures for establishing, executing, and interpreting AFS and TIRF measurements aimed at exploring DNA compaction driven by TFAM.

Mitochondrial DNA, or mtDNA, is housed within nucleoid structures, a characteristic feature of these organelles. In situ visualization of nucleoids is possible with fluorescence microscopy, but the introduction of stimulated emission depletion (STED) super-resolution microscopy has opened the door to sub-diffraction resolution visualization of nucleoids.