Mitochondrial DNA has been investigated for nearly fifty years, but many aspects of the maintenance of this essential small genome remain unknown. Like any genome, mammalian mitochondrial DNA requires the function of topoisomerases to counter and regulate the topological tension arising during replication, transcription, segregation, and repair. However, the functions of the different mitochondrial topoisomerases are poorly understood. Here, we investigate the role of Topoisomerase 3 alpha (Top3 alpha) in mtDNA replication and transcription, providing evidence that this enzyme, previously reported to act in mtDNA segregation, also participates in mtDNA replication fork progression. Top3 alpha knockdown caused replication fork stalling, increased mtDNA catenation and decreased mtDNA levels. Overexpression in contrast induced abundant double-strand breaks around the replication origin O-H and abortion of early replication, while at the same time improving the resolution of mtDNA replication termination intermediates. Both Top3 alpha knockdown and overexpression affected mitochondrial RNA transcription, leading to a decrease in steady-state levels of mitochondrial transcripts. Together, our results indicate that the mitochondrial isoform of Top3 alpha is not only involved in mtDNA segregation, as reported previously, but also supports the progression of the replication fork. Mitochondrial Top3 alpha is also influencing the progression of transcription, with its absence affecting downstream transcript levels.
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