Fig 1: Topoisomerase 2a and 2ß co-localize with replicating mitochondrial DNA nucleoids. (A) Detection of topoisomerases in mitochondrial extracts of human and mouse cell lines by Western blot. Human cell lines: HEK293T and HeLa. Mouse cells: proliferating C2C12 myoblasts and differentiated C2C12 myotubes, mouse embryonic fibroblasts of wildtype and Top2ß (–/–) knockout mice. ATAD3 is used as loading reference. Note that the used polyclonal antibodies might detect mouse and human proteins with different efficiencies. (B) Detection of topoisomerase Top2ß, Top1mt and Top3a in mitochondrial extracts of various mouse tissues by Western blot. TFAM is used as indicator of mtDNA content, while TOMM20 serves as mitochondrial loading control. (C) Localization of mitochondrial topoisomerases in floatation gradients of mitochondrial extracts from HEK293T cells. The pellet fraction after digitonin treatment contains inner membrane-associated proteins and nucleoids, while the supernatant fraction contains soluble nucleoids and proteins without membrane-association. Top2a and Top2b co-localize well with the membrane-attached, replicating mtDNA nucleoids characterized by the presence of the mitochondrial helicase TWNK. Also the mitochondrial topoisomerases Top3a and Top1mt partially co-localize with this fraction, but a larger proportion is found in other fractions. (D) Mitochondrially targeted Top2ß co-localizes with replicating nucleoids. HeLa cells transiently transfected with an expression vector for myc-tagged, mitochondrially targeted Top2ß were immuno-stained with a monoclonal antibody against the myc-tag (green) and a polyclonal antibody against mtSSB (red) as a marker for mtDNA replication.