Several human pathologies are characterized by the presence of mitochondrial DNA (mtDNA) mutations, which are also connected to the aging process. Essential genes for mitochondrial function are absent due to deletion mutations within the mitochondrial DNA. 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. Forty-nine hundred and seventy-seven base pairs of mtDNA are eliminated by this deletion. The formation of the commonplace deletion has been previously shown to be influenced by exposure to UVA radiation. Subsequently, inconsistencies in mitochondrial DNA replication and repair procedures are connected to the production of the prevalent deletion. Nonetheless, the molecular mechanisms underlying this deletion's formation remain poorly understood. To detect the common deletion in human skin fibroblasts, this chapter details a method involving irradiation with physiological doses of UVA, and subsequent quantitative PCR analysis.

Deoxyribonucleoside triphosphate (dNTP) metabolism abnormalities can contribute to the development of mitochondrial DNA (mtDNA) depletion syndromes (MDS). The muscles, liver, and brain are compromised by these disorders, where the concentrations of dNTPs in those tissues are naturally low, which makes the process of measurement difficult. Accordingly, information regarding the concentrations of dNTPs in the tissues of animals without disease and those suffering from MDS holds significant importance for understanding the mechanisms of mtDNA replication, monitoring disease development, and developing therapeutic strategies. This study details a sophisticated technique for the simultaneous measurement of all four dNTPs and all four ribonucleoside triphosphates (NTPs) in mouse muscle, achieved by employing hydrophilic interaction liquid chromatography and triple quadrupole mass spectrometry. The concurrent discovery of NTPs allows their employment as internal reference points for the standardization of dNTP concentrations. In other tissues and organisms, this method can be used to measure the presence of dNTP and NTP pools.

Two-dimensional neutral/neutral agarose gel electrophoresis (2D-AGE) has been employed in the study of animal mitochondrial DNA replication and maintenance for nearly two decades, but its potential remains largely unrealized. We outline the steps in this procedure, from DNA extraction, through two-dimensional neutral/neutral agarose gel electrophoresis and subsequent Southern hybridization, to the final interpretation of the results. We additionally present instances of 2D-AGE's application in examining the diverse characteristics of mtDNA maintenance and regulation.

Employing substances that disrupt DNA replication to modify mitochondrial DNA (mtDNA) copy number in cultured cells provides a valuable method for exploring diverse facets of mtDNA maintenance. Our study describes how 2',3'-dideoxycytidine (ddC) can reversibly decrease the copy number of mitochondrial DNA (mtDNA) 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 enzymatic activity of the mtDNA replication machinery is valuably assessed through the dynamics of mtDNA repopulation.

Endosymbiotic in origin, eukaryotic mitochondria possess their own genetic code, mitochondrial DNA, and mechanisms dedicated to the DNA's maintenance and expression. The mitochondrial oxidative phosphorylation system necessitates all proteins encoded by mtDNA molecules, despite the limited count of such proteins. This report outlines protocols for observing DNA and RNA synthesis processes in intact, isolated mitochondria. Mechanisms of mtDNA maintenance and expression regulation can be effectively studied using organello synthesis protocols as powerful tools.

The accurate duplication of mitochondrial DNA (mtDNA) is fundamental to the proper operation of the cellular 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. To study how the mtDNA replisome responds to oxidative or UV-damaged DNA, an in vitro reconstituted mtDNA replication system is a viable approach. The methodology for studying DNA damage bypass, employing a rolling circle replication assay, is meticulously detailed in this chapter. This assay, built on purified recombinant proteins, is adaptable for investigating various aspects of mitochondrial DNA (mtDNA) preservation.

TWINKLE, an indispensable helicase, is responsible for the unwinding of the mitochondrial genome's duplex DNA during the DNA replication process. In vitro assays involving purified recombinant forms of the protein have been critical for gaining mechanistic understanding of the function of TWINKLE at the replication fork. We present methods to study the helicase and ATPase activities exhibited by TWINKLE. In order to perform the helicase assay, TWINKLE is incubated with a radiolabeled oligonucleotide that has been annealed to a single-stranded M13mp18 DNA template. The oligonucleotide, subsequently visualized via gel electrophoresis and autoradiography, will be displaced by TWINKLE. The release of phosphate, a consequence of TWINKLE's ATP hydrolysis, is precisely quantified using a colorimetric assay, thereby measuring the enzyme's ATPase activity.

Inherent to their evolutionary origins, mitochondria include their own genome (mtDNA), condensed into the mitochondrial chromosome or the nucleoid (mt-nucleoid). Disruptions of mt-nucleoids frequently present in mitochondrial disorders, due to either direct mutations in genes regulating mtDNA organization or interference with other crucial proteins necessary for mitochondrial functions. infections after HSCT Thusly, changes in the mt-nucleoid's morphology, dissemination, and composition are frequently present in various human maladies, and they can be exploited to assess cellular proficiency. Electron microscopy is instrumental in reaching the highest resolution possible, providing information on the spatial structure of every cellular component. Employing ascorbate peroxidase APEX2, recent studies have sought to enhance transmission electron microscopy (TEM) contrast through the process of inducing 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. 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. When hydrogen peroxide is present, APEX2 catalyzes the polymerization of DAB, forming a brown precipitate that can be visualized within specific areas of the mitochondrial matrix. To visualize and target mt-nucleoids, we detail a protocol for creating murine cell lines expressing a transgenic Twinkle variant. In addition, we delineate every crucial step in validating cell lines before electron microscopy imaging, along with examples of expected results.

MtDNA's replication and transcription processes take place in the compact nucleoprotein complexes of mitochondrial nucleoids. 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. We delineate a proximity-biotinylation assay, BioID, enabling the identification of proteins closely interacting with mitochondrial nucleoid proteins. Biotin is covalently attached to lysine residues on neighboring proteins by a promiscuous biotin ligase fused to the protein of interest. Biotin-affinity purification procedures can be applied to enrich biotinylated proteins for subsequent identification by mass spectrometry. Transient and weak interactions are discernible using BioID, allowing for the identification of alterations in these interactions under diverse cellular treatment regimens, different protein isoforms, or pathogenic variants.

The protein mitochondrial transcription factor A (TFAM), essential for mtDNA, binds to it to initiate mitochondrial transcription and maintain its integrity. Considering TFAM's direct interaction with mitochondrial DNA, understanding its DNA-binding capacity proves helpful. 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 effects of mutations, truncation, and post-translational modifications on the function of this essential mtDNA regulatory protein are explored using these instruments.

Mitochondrial transcription factor A (TFAM) orchestrates the arrangement and compactness of the mitochondrial genome. CSF biomarkers Although there are constraints, only a small number of simple and readily achievable methodologies are available for monitoring and quantifying TFAM's influence on DNA condensation. The straightforward single-molecule force spectroscopy technique, Acoustic Force Spectroscopy (AFS), employs acoustic methods. 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. Ki16198 nmr This document meticulously details the setup, execution, and analysis of AFS and TIRF measurements, with a focus on comprehending how TFAM affects DNA compaction.

The DNA within mitochondria, specifically mtDNA, is compactly packaged inside structures known as nucleoids. While in situ visualization of nucleoids is achievable through fluorescence microscopy, stimulated emission depletion (STED) super-resolution microscopy has enabled a more detailed view of nucleoids, resolving them at sub-diffraction scales.