Article

Analysis of mitochondrial DNA by two-dimensional agarose gel electrophoresis.

MRC Mitochondrial Biology Unit, Wellcome Trust/MRC Building, Cambridge, UK.
Methods in molecular biology (Clifton, N.J.) 02/2009; 554:15-35. DOI:10.1007/978-1-59745-521-3_2 pp.15-35
Source: PubMed

ABSTRACT 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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    Article: Mitochondrial DNA replication proceeds via a ‘bootlace’ mechanism involving the incorporation of processed transcripts
    Aurelio Reyes, Lawrence Kazak, Stuart R. Wood, Takehiro Yasukawa, Howard T. Jacobs, Ian J. Holt
    [show abstract] [hide abstract]
    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.
    Nucleic Acids Research 04/2013; 41(7). · 8.03 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.

Keywords

circular molecules
 
derive information
 
DNA fragments
 
DNA hybrid
 
DNA replication
 
DNA-binding proteins
 
higher vertebrates
 
intact mitochondrial replication intermediates
 
mitochondria
 
mitochondrial DNA
 
mitochondrial nucleoprotein
 
mtDNA
 
multigenomic nucleoprotein complexes
 
nucleoid superstructure
 
nucleoids
 
replication
 
Replication intermediates
 
segregation
 
solid tissues
 
twin processes