Analysis of Mitochondrial DNA Replication by Two-Dimensional Agarose Gel Electrophoresis

Goffart, Steffi; Pohjoismäki, Jaakko L. O.

doi:10.1007/978-1-0716-2922-2_18

Cited by 1 publication

(1 citation statement)

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“…MtDNA topology and replication intermediates were analyzed by one- and two-dimensional agarose gel electrophoresis and Southern blotting (Goffart and Pohjoismaki, 2023). Briefly, cells were harvested, washed once with 1 x PBS and pelleted by centrifugation at 400 g for 4 minutes.…”

Section: Methodsmentioning

confidence: 99%

A small molecule inhibitor Mirin prevents TOP3A-dependent mtDNA breakage and segregation

Aasumets,

Hangas,

Bader

et al. 2024

Preprint

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Mirin, the chemical inhibitor of MRE11, has been recently reported to prevent immune response activation caused by mitochondrial DNA (mtDNA) breakage and release upon replication stalling. We show here that Mirin prevents mitochondrial replication fork breakage in mitochondrial 3'-exonuclease MGME1 deficient cells and the resulting innate immune response induction, but that this occurs independently of MRE11. Furthermore, Mirin also caused alteration of mtDNA supercoiling and accumulation of hemicatenated replication termination intermediates, hallmarks of topoisomerase dysfunction, as well as alleviated topological changes induced by the overexpression of mitochondrial TOP3A, including TOP3A-dependent strand breakage at the non-coding region of mtDNA, potentially explaining its protective effect in the MGME1-knockout cells. Although Mirin does not inhibit TOP3A in vitro, our results demonstrate its MRE11-independent effects in cells and give insight into the mechanisms of mtDNA segregation, as well as the maintenance of genomic integrity in mitochondria.

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Section: Methodsmentioning

confidence: 99%