Analysis of Mitochondrial DNA by Two-Dimensional Agarose Gel Electrophoresis

MRC Mitochondrial Biology Unit, Wellcome Trust/MRC Building, Cambridge, UK.
Methods in Molecular Biology (Impact Factor: 1.29). 02/2009; 554:15-35. DOI: 10.1007/978-1-59745-521-3_2
Source: PubMed


In higher vertebrates, the DNA of mitochondria takes the form of circular molecules of approximately 16 kbp. These circles are arranged in multigenomic nucleoprotein complexes or nucleoids. It is envisaged that nucleoid superstructure makes a critical contribution to the twin processes of replication and segregation of mtDNA. Replication intermediates can be isolated from cells or solid tissues and separated on agarose gels in two dimensions to reveal a wealth of data on mechanisms of DNA replication. Using this technique we have demonstrated that many molecules of replicating mtDNA have extensive regions of RNA: DNA hybrid in higher vertebrates. More recently, we have extracted mitochondrial nucleoprotein and analyzed it by the same method to derive information on the distribution of DNA-binding proteins on mitochondrial DNA. Here we describe the procedures used to isolate intact mitochondrial replication intermediates from liver and cultured cells of higher vertebrates and the process of separating DNA fragments on neutral two-dimensional agarose gels.

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    • "mtDNA from sucrose-gradient purified mitochondria was prepared as described previously (12), except that isolated mitochondria were treated with Proteinase K at 4°C for 10–45 min before detergent lysis and successive phenol and chloroform/isoamylalcohol (24:1) extractions. In some cases, whole tissue DNA was extracted directly from homogenized rat liver using phenol and chloroform, after being solubilized with 1% sodium N-lauroylsarcosinate and incubated with 100 µg/ml Proteinase K on ice for 30 min. "
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    ABSTRACT: The observation that long tracts of RNA are associated with replicating molecules of mitochondrial DNA (mtDNA) suggests that the mitochondrial genome of mammals is copied by an unorthodox mechanism. Here we show that these RNA-containing species are present in living cells and tissue, based on interstrand cross-linking. Using DNA synthesis in organello, we demonstrate that isolated mitochondria incorporate radiolabeled RNA precursors, as well as DNA precursors, into replicating DNA molecules. RNA-containing replication intermediates are chased into mature mtDNA, to which they are thus in precursor–product relationship. While a DNA chain terminator rapidly blocks the labeling of mitochondrial replication intermediates, an RNA chain terminator does not. Furthermore, processed L-strand transcripts can be recovered from gel-extracted mtDNA replication intermediates. Therefore, instead of concurrent DNA and RNA synthesis, respectively, on the leading and lagging strands, preformed processed RNA is incorporated as a provisional lagging strand during mtDNA replication. These findings indicate that RITOLS is a physiological mechanism of mtDNA replication, and that it involves a ‘bootlace' mechanism, in which processed transcripts are successively hybridized to the lagging-strand template, as the replication fork advances.
    Full-text · Article · Apr 2013 · Nucleic Acids Research
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    • "Up until that point, Y (including broken bubble) and dY structures migrate very close together. Unfortunately, the region between the standard Y and X arcs where dYs will migrate is also very compressed, due to the gel-running conditions [22], [23] that must be employed to resolve fragments of the size of the linearized mitochondrial genome (15–20 kb). Multiple, less prominent intermediates are visible migrating below the Y arc after S1 nuclease treatment (e.g. Figure 2). "
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    ABSTRACT: Mitochondrial DNA synthesis is necessary for the normal function of the organelle and for the eukaryotic organism as a whole. Here we demonstrate, using two-dimensional agarose gel electrophoresis to analyse replication intermediates, that unidirectional, strand-coupled DNA synthesis is the prevalent mode of mtDNA replication in Drosophila melanogaster. Commencing within the single, extended non-coding region (NCR), replication proceeds around the circular genome, manifesting an irregular rate of elongation, and pausing frequently in specific regions. Evidence for a limited contribution of strand-asynchronous DNA synthesis was found in a subset of mtDNA molecules, but confined to the ribosomal RNA gene region, just downstream of the NCR. Our findings imply that strand-coupled replication is widespread amongst metazoans, and should inform future research on mtDNA metabolism in D. melanogaster.
    Preview · Article · Jan 2013 · PLoS ONE
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    • "The addition of a second gel separation step, which uses ethidium bromide (EtBr) intercalation to compact and accelerate topologically confined DNA, can further resolve undigested genomes, but it has only been applied to petite yeast mtDNA (29) and high-molecular weight structures in heart (27). This approach contrasts the classic 2D agarose electrophoresis gels (2D-AGE) used to examine fragments of genomes (30). In the present study, we adapted the concept of resolving undigested (intact) mtDNA to optimize the separation of all major structures, a technique that we call 2D intact mitochondrial DNA agarose gel electrophoresis (2D-IMAGE). "
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    ABSTRACT: The mitochondrial genome exists in numerous structural conformations, complicating the study of mitochondrial DNA (mtDNA) metabolism. Here, we describe the development of 2D intact mtDNA agarose gel electrophoresis (2D-IMAGE) for the separation and detection of approximately two-dozen distinct topoisomers. Although the major topoisomers were well conserved across many cell and tissue types, unique differences in certain cells and tissues were also observed. RNase treatment revealed that partially hybridized RNAs associated primarily with covalently closed circular DNA, consistent with this structure being the template for transcription. Circular structures composed of RNA:DNA hybrids contained only heavy-strand DNA sequences, implicating them as lagging-strand replication intermediates. During recovery from replicative arrest, 2D-IMAGE showed changes in both template selection and replication products. These studies suggest that discrete topoisomers are associated with specific mtDNA-directed processes. Because of the increased resolution, 2D-IMAGE has the potential to identify novel mtDNA intermediates involved in replication or transcription, or pathology including oxidative linearization, deletions or depletion.
    Full-text · Article · Dec 2012 · Nucleic Acids Research
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