Several human pathologies are characterized by the presence of mitochondrial DNA (mtDNA) mutations, which are also connected to the aging process. Genetic deletions within mitochondrial DNA diminish the availability of necessary genes critical for mitochondrial function. Over 250 deletion mutations have been observed in the literature, and the most frequent mtDNA deletion is commonly linked to disease conditions. This deletion operation removes a segment of mtDNA, containing precisely 4977 base pairs. UVA radiation has been previously shown to encourage the formation of the frequently occurring deletion. Concerningly, variations in mtDNA replication and repair are factors in the occurrence of the common deletion. In contrast, the molecular mechanisms governing this deletion's formation are poorly characterized. The chapter outlines a procedure for exposing human skin fibroblasts to physiological UVA doses, culminating in the quantitative PCR detection of the frequent deletion.
Problems in the deoxyribonucleoside triphosphate (dNTP) metabolic process are frequently observed in cases of mitochondrial DNA (mtDNA) depletion syndromes (MDS). These disorders manifest in the muscles, liver, and brain, where dNTP concentrations are intrinsically low in the affected tissues, complicating measurement. Hence, the concentrations of dNTPs in the tissues of both healthy and myelodysplastic syndrome (MDS) animals are vital for mechanistic examinations of mitochondrial DNA (mtDNA) replication, tracking disease progression, and developing therapeutic interventions. 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. The simultaneous observation of NTPs allows them to function as internal controls for the standardization of dNTP quantities. Measuring dNTP and NTP pools in other tissues and organisms is facilitated by this applicable method.
In the study of animal mitochondrial DNA replication and maintenance processes, two-dimensional neutral/neutral agarose gel electrophoresis (2D-AGE) has been employed for nearly two decades; however, its full capabilities remain largely untapped. From the initial DNA isolation process to the subsequent two-dimensional neutral/neutral agarose gel electrophoresis, the subsequent Southern blot hybridization, and the conclusive data analysis, we detail the procedure. Furthermore, we illustrate how 2D-AGE can be utilized to explore the various aspects of mtDNA upkeep and control.
A useful means of exploring diverse aspects of mtDNA maintenance is the manipulation of mitochondrial DNA (mtDNA) copy number in cultured cells via the application of substances that impair 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. When ddC application ceases, cells with diminished mtDNA levels strive to recover their usual mtDNA copy count. The process of mtDNA repopulation dynamically reflects the enzymatic efficiency of the mtDNA replication system.
Mitochondrial DNA (mtDNA) is present in eukaryotic mitochondria which have endosymbiotic origins and are accompanied by systems dedicated to its care and expression. Even though the number of proteins encoded by mtDNA molecules is restricted, they are all critical elements of the mitochondrial oxidative phosphorylation pathway. Intact, isolated mitochondria are the subject of the protocols described here for monitoring DNA and RNA synthesis. Organello synthesis protocols are essential techniques for examining the regulatory mechanisms and processes governing mtDNA maintenance and expression.
Mitochondrial DNA (mtDNA) replication's integrity is vital for the proper performance of the oxidative phosphorylation system. 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. 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. For the assay, purified recombinant proteins provide the foundation, and it can be adjusted to analyze multiple facets of mtDNA preservation.
The helicase TWINKLE is indispensable for the task of unwinding the mitochondrial genome's double-stranded structure during DNA replication. For gaining mechanistic insights into the role of TWINKLE at the replication fork, in vitro assays using purified recombinant proteins have been essential tools. Our approach to investigating TWINKLE's helicase and ATPase functions is outlined here. A radiolabeled oligonucleotide, annealed to an M13mp18 single-stranded DNA template, is incubated with TWINKLE for the helicase assay. Using gel electrophoresis and autoradiography, the oligonucleotide, displaced by TWINKLE, is visualized. Quantifying the phosphate release resulting from ATP hydrolysis by TWINKLE is accomplished using a colorimetric assay, which then measures the ATPase activity.
Bearing a resemblance to their evolutionary origins, mitochondria possess their own genetic material (mtDNA), condensed into the mitochondrial chromosome or 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. find more Consequently, alterations in mt-nucleoid morphology, distribution, and structure are frequently observed in various human ailments and can serve as a marker for cellular vitality. Electron microscopy offers the highest attainable resolution, enabling the precise visualization and understanding of the spatial arrangement and structure 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, accumulating within DAB during classical electron microscopy sample preparation, affords strong contrast in transmission electron microscopy images due to the substance's 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. H2O2 activates APEX2's function in DAB polymerization, creating a detectable brown precipitate within particular compartments of the mitochondrial matrix. We furnish a thorough method for creating murine cell lines that express a genetically modified version of Twinkle, enabling the targeting and visualization 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, compact nucleoprotein complexes, house, replicate, and transcribe mtDNA. Previous efforts in proteomic analysis to identify nucleoid proteins have been undertaken; however, a definitive list of nucleoid-associated proteins has not been compiled. This proximity-biotinylation assay, BioID, is described here, facilitating the identification of nearby proteins associated with mitochondrial nucleoid proteins. The protein of interest, bearing a promiscuous biotin ligase, establishes covalent biotin linkages with lysine residues on its neighboring proteins. Proteins tagged with biotin can be subjected to further enrichment through biotin-affinity purification, followed by mass spectrometry identification. BioID allows the identification of both transient and weak interactions, and further allows for the assessment of modifications to these interactions induced by diverse cellular manipulations, protein isoform alterations, or pathogenic variations.
Mitochondrial transcription factor A (TFAM), a mtDNA-binding protein, facilitates mitochondrial transcription initiation and, concurrently, supports mtDNA maintenance. 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. These tools are utilized to explore how mutations, truncation, and post-translational modifications influence the function of this crucial mtDNA regulatory protein.
A key function of mitochondrial transcription factor A (TFAM) is the organization and condensation of the mitochondrial genome. find more Yet, a restricted number of simple and accessible techniques are available for quantifying and observing the DNA compaction that TFAM is responsible for. The single-molecule force spectroscopy technique known as Acoustic Force Spectroscopy (AFS) is straightforward. The system facilitates the simultaneous tracking of multiple individual protein-DNA complexes, allowing for the determination of 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. find more 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.
Mitochondria possess their own genetic material, mtDNA, organized within nucleoid structures. In situ nucleoid visualization is possible via fluorescence microscopy; however, the introduction of super-resolution microscopy, particularly stimulated emission depletion (STED), enables viewing nucleoids at a sub-diffraction resolution.