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 3α (Top3α) in mtDNA replication and transcription, providing evidence that this enzyme, previously reported to act in mtDNA segregation, also participates in mtDNA replication fork progression. Top3α knockdown caused replication fork stalling, increased mtDNA catenation and decreased mtDNA levels. Overexpression in contrast induced abundant double-strand breaks around the replication origin OH and abortion of early replication, while at the same time improving the resolution of mtDNA replication termination intermediates. Both Top3α 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 Top3α is not only involved in mtDNA segregation, as reported previously, but also supports the progression of the replication fork. Mitochondrial Top3α is also influencing the progression of transcription, with its absence affecting downstream transcript levels.

中文翻译：

---

Top3α是线粒体DNA复制中的复制拓扑异构酶

线粒体 DNA 已被研究了近 50 年，但维持这一重要的小基因组的许多方面仍然未知。与任何基因组一样，哺乳动物线粒体 DNA 需要拓扑异构酶的功能来对抗和调节复制、转录、分离和修复过程中产生的拓扑张力。然而，人们对不同线粒体拓扑异构酶的功能知之甚少。在这里，我们研究了拓扑异构酶 3α (Top3α) 在 mtDNA 复制和转录中的作用，提供了证据表明这种酶，以前报道过在 mtDNA 分离中起作用，也参与了 mtDNA 复制叉的进程。Top3α 敲低导致复制叉停止，增加 mtDNA 连接并降低 mtDNA 水平。相反，过表达会在复制起点 OH 周围诱导大量双链断裂和早期复制的中止，同时提高 mtDNA 复制终止中间体的分辨率。Top3α 敲低和过表达都会影响线粒体 RNA 转录，导致线粒体转录物的稳态水平下降。总之，我们的结果表明，Top3α 的线粒体同种型不仅参与 mtDNA 分离，如前所述，而且还支持复制叉的进展。线粒体 Top3α 也影响转录进程，其缺失会影响下游转录水平。同时提高 mtDNA 复制终止中间体的分辨率。Top3α 敲低和过表达都会影响线粒体 RNA 转录，导致线粒体转录物的稳态水平下降。总之，我们的结果表明，Top3α 的线粒体同种型不仅参与 mtDNA 分离，如前所述，而且还支持复制叉的进展。线粒体 Top3α 也影响转录进程，其缺失会影响下游转录水平。同时提高 mtDNA 复制终止中间体的分辨率。Top3α 敲低和过表达都会影响线粒体 RNA 转录，导致线粒体转录物的稳态水平下降。总之，我们的结果表明，Top3α 的线粒体同种型不仅参与 mtDNA 分离，如前所述，而且还支持复制叉的进展。线粒体 Top3α 也影响转录进程，其缺失会影响下游转录水平。如前所述，但也支持复制叉的进展。线粒体 Top3α 也影响转录进程，其缺失会影响下游转录水平。如前所述，但也支持复制叉的进展。线粒体 Top3α 也影响转录进程，其缺失会影响下游转录水平。