Human pathologies frequently display the presence of mitochondrial DNA (mtDNA) mutations, a characteristic also associated with aging. Mitochondrial DNA deletion mutations are responsible for the removal of essential genes, consequently affecting 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 process eliminates 4977 base pairs from the mtDNA sequence. Prior research has exhibited that UVA light exposure can stimulate the production of the prevalent deletion. Additionally, deviations in mtDNA replication and repair mechanisms contribute to the formation of the common deletion. Yet, the molecular mechanisms responsible for the development of this deletion 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.
Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are characterized by defects in the metabolism of deoxyribonucleoside triphosphate (dNTP). The muscles, liver, and brain are affected by these disorders, and the dNTP concentrations in these tissues are already naturally low, thus making measurement challenging. In this manner, details on dNTP concentrations in healthy and myelodysplastic syndrome (MDS)-afflicted animal tissues are essential for mechanistic investigations into mtDNA replication, an assessment of disease progression, and the design of therapeutic approaches. A sensitive approach for the simultaneous quantification of all four dNTPs and all four ribonucleoside triphosphates (NTPs) in mouse muscle is detailed, utilizing hydrophilic interaction liquid chromatography in conjunction with triple quadrupole mass spectrometry. The simultaneous observation of NTPs allows them to function as internal controls for the standardization of dNTP quantities. The method's utility encompasses the measurement of dNTP and NTP pools in a wide spectrum of tissues and organisms.
The application of two-dimensional neutral/neutral agarose gel electrophoresis (2D-AGE) in studying animal mitochondrial DNA replication and maintenance processes has continued for almost two decades, though the method's full potential has not been fully explored. 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. Along with our analysis, we provide examples of how 2D-AGE analysis can be used to explore the multifaceted nature 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. 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. Following the discontinuation of ddC administration, cells exhibiting mtDNA depletion seek to regain their standard mtDNA copy numbers. Mitochondrial DNA (mtDNA) repopulation kinetics serve as a significant indicator of the enzymatic activity inherent in the mtDNA replication apparatus.
Mitochondrial DNA (mtDNA) is present in eukaryotic mitochondria which have endosymbiotic origins and are accompanied by systems dedicated to its care and expression. A constrained number of proteins are encoded within mtDNA molecules, yet every one of these proteins is an indispensable element of the mitochondrial oxidative phosphorylation complex. Procedures for monitoring DNA and RNA synthesis in intact, isolated mitochondria are described in the following protocols. The application of organello synthesis protocols is critical for the study of mtDNA maintenance and its expression mechanisms and regulatory processes.
Accurate mitochondrial DNA (mtDNA) replication is indispensable for the correct functioning of the 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. The mechanisms by which the mtDNA replisome addresses oxidative or ultraviolet DNA damage can be explored using a reconstituted mtDNA replication system in a test tube. We elaborate, in this chapter, a detailed protocol for exploring the bypass of diverse DNA damages via a rolling circle replication assay. The assay's capability rests on purified recombinant proteins and it can be adjusted to the investigation of different aspects of mtDNA maintenance.
Helicase TWINKLE is crucial for unwinding the mitochondrial genome's double helix during DNA replication. 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. This paper demonstrates methods for characterizing the helicase and ATPase properties of TWINKLE. The helicase assay protocol entails the incubation of TWINKLE with a radiolabeled oligonucleotide that is hybridized to a single-stranded M13mp18 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.
Due to their evolutionary lineage, mitochondria contain their own genetic material (mtDNA), compressed into the mitochondrial chromosome or the nucleoid (mt-nucleoid). Mitochondrial disorders often exhibit disruptions in mt-nucleoids, stemming from either direct mutations in genes associated with mtDNA organization or interference with essential mitochondrial proteins. Infectious Agents Accordingly, changes to mt-nucleoid form, spread, and arrangement are a common characteristic of many human illnesses and can be employed to assess cellular well-being. All cellular structures' spatial and structural properties are elucidated through electron microscopy's unique ability to achieve the highest possible resolution. A novel approach to increasing contrast in transmission electron microscopy (TEM) images involves the use of ascorbate peroxidase APEX2 to induce the precipitation of diaminobenzidine (DAB). The ability of DAB to accumulate osmium during classical electron microscopy sample preparation contributes to its high electron density, thereby producing strong contrast in transmission electron microscopy. To visualize mt-nucleoids with high contrast and electron microscope resolution, a tool utilizing the fusion of mitochondrial helicase Twinkle with APEX2 has been successfully implemented among nucleoid proteins. 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. Prior to electron microscopy imaging, we also provide a comprehensive explanation of the necessary steps for validating cell lines, illustrated by examples of expected outcomes.
Within mitochondrial nucleoids, the compact nucleoprotein complexes are the sites for the replication and transcription of 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 document details the proximity-biotinylation assay, BioID, which facilitates the identification of mitochondrial nucleoid protein interaction partners. The protein of interest, bearing a promiscuous biotin ligase, establishes covalent biotin linkages with lysine residues on its neighboring proteins. Mass spectrometry analysis can identify biotinylated proteins after their enrichment via a biotin-affinity purification process. BioID possesses the capability to identify both transient and weak protein-protein interactions, and it can further be utilized to determine any changes to these interactions under different cellular treatments, protein isoforms or pathogenic forms.
Mitochondrial transcription factor A (TFAM), a protein that binds mitochondrial DNA (mtDNA), undertakes a dual function, initiating mitochondrial transcription and upholding mtDNA stability. Because of TFAM's direct connection to mtDNA, examining its DNA-binding capabilities provides useful data. In this chapter, two in vitro assay methods, an electrophoretic mobility shift assay (EMSA) and a DNA-unwinding assay, are described. Both utilize recombinant TFAM proteins and are contingent on the employment of simple agarose gel electrophoresis. Investigations into the effects of mutations, truncations, and post-translational modifications on this vital mtDNA regulatory protein are conducted using these tools.
Mitochondrial transcription factor A (TFAM) actively participates in the arrangement and compression of the mitochondrial genetic material. AUNP12 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. Acoustic Force Spectroscopy (AFS), a straightforward method, facilitates single-molecule force spectroscopy. Parallel tracking of numerous individual protein-DNA complexes is facilitated, allowing for the quantification of their mechanical properties. The dynamics of TFAM's interactions with DNA in real time are revealed by the high-throughput single-molecule approach of TIRF microscopy, a capability not offered by traditional biochemistry methods. medium spiny neurons 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. 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.