Euromit 2017

These are the projects we are planning to present in our next conference in Cologne (Germany): Euromit 2017.

Mitochondrial Topoisomerases (Anu hangas)

Topoisomerases are essential enzymes for replication, transcription and repair of DNA in any known organism as they regulate the supercoiling and winding of DNA molecules. While their function is rather well characterized in nuclear DNA metabolism, surprisingly little is known about topoisomerases in mitochondria, where the constant expression and replication of mitochondrial DNA equally requires their function. Information about those enzymes is urgently needed as topoisomerase inhibitors are broadly used, e.g. in cancer therapy or as antibiotics often having severe side effects.

To address the function of Top2β in mitochondria, we studied mitochondrial DNA alterations upon knockdown of Top2β. In addition, we investigated the effects of a suspected topoisomerase 2 inhibitor, the commonly used antibiotic ciprofloxacin, on mitochondrial DNA maintenance in proliferating HeLa cells and in differentiated C2C12 cells.

Role of PrimPol in mtDNA replication (Rubén Torregrosa-Muñumer)

During the mitochondrial DNA (mtDNA) replication, replication fork can stall due to damaged DNA template or deleterious mutations in enzymes required for mtDNA maintenance. The stalled replication forks can collapse, resulting in double-strand breaks and the loss of partially replicated genomes, being a potential cause of the pathological mtDNA deletions seen in human diseases. Replication stalling occurs also in the nucleus, where multiple pathways are involved in replication fork resolution, rescue and re-initiation. However, the fate of stalled replication forks in mitochondria is unclear. To make even more complicated the understanding of mtDNA maintenance, there are indications suggesting that at least two mechanistically different mtDNA replication mechanisms operate in mammals, the so-called RITOLS and COSCOFA. Interestingly, oxidative and UV damage seems to specifically stall RITOLS intermediates and induce COSCOFA replication. It might be that RITOLS represents a high fidelity, housekeeping mode, whereas COSCOFA is a more robust replication mechanism operating under stress.

Figure 1 – mtDNA replication fork stalling: during mtDNA replication, the mtDNA replication fork can stall due to damaged DNA template, mutated mtDNA maintenance proteins or inhibitors of the mitochondrial DNA polymerase, among others. Prolonged mtDNA stalling can lead to fork collapse and result in rearrangements and deletions, with dramatic consequences for the mitochondrial function. However, nothing is known about the fate of mtDNA stalled replication forks. In the nucleus, fork collapse can be resolved by double strand break formation and repair or fork regression to restart of replication. In another scenario, PrimPol has risen as a potential re-priming enzyme, which seems to allow replication restart downstream the lesion.

For the last few years our research group has been trying to understand the interplay between replicative stress and replication mode switching in mitochondria. Our investigations have concentrated on recently discovered primase-polymerase enzyme PrimPol, which is located both in the nucleus and in the mitochondria. Although PrimPol was firstly identified as a translesion polymerase, able to bypass damage on the DNA template, together with some other recent studies our results give more importance to the priming activity of PrimPol, including the priming of lagging-strand replication at unconventional origins on mtDNA. We show for the first time that PrimPol, although it is not an essential enzyme for mtDNA maintenance, it plays a significant role in maintaining replication under genotoxic stress and possibly explaining some of key features of the different replication mechanisms.

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