Abstract
Human mitochondrial DNA is a small circular double-stranded molecule that is essential for cellular energy production. A specialized protein machinery replicates the mitochondrial genome, with DNA polymerase γ carrying out synthesis of both strands. According to the prevailing mitochondrial DNA replication model, the two strands are replicated asynchronously, with the leading heavy-strand initiating first, followed by the lagging light-strand. By using purified recombinant forms of the replication proteins and synthetic DNA templates, it is possible to reconstitute mitochondrial DNA replication in vitro. Here we provide details on how to differentially reconstitute replication of the leading- and lagging-strands.
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Acknowledgements
We thank lab members for their input, especially Majda Mehmedovic and Emily Hoberg for experimental demonstrations, and Örjan Persson, Yazh Muthukumar, and Hector Diaz for technical input. This work was supported by grants to MF from the Swedish Research Council; Swedish Cancer Foundation; European Research Council; the IngaBritt and Arne Lundberg Foundation; and the Knut and Alice Wallenberg Foundation.
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Uhler, J.P., Falkenberg, M. (2021). In Vitro Analysis of mtDNA Replication. In: Minczuk, M., Rorbach, J. (eds) Mitochondrial Gene Expression. Methods in Molecular Biology, vol 2192. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0834-0_1
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DOI: https://doi.org/10.1007/978-1-0716-0834-0_1
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