Introduction
The overall aim of this research project is to study the mechanisms of mitochondrial mtDNA maintenance in mammalian cells. The initial phase of the project has been very successful and a number of findings have been published or are in the pipeline for future publications. Please see below a short description of the different aims in the initial application and the results achieved so far.
Aim 1. To define how the borders of the mitochondrial D-loop is formed.
The function of the D-loop and how the formation of this structure is regulated is poorly understood. We have in yet to be published experiments demonstrated the existence of a new transcriptional start site within the D-loop region and we are currently characterizing the function of this site in vitro and in vivo. In another set of experiments we have found that SUV3, a RNA/DNA helicase is important for D-loop metabolism and mtDNA maintenance. Our findings have revealed that SUV3 plays an important role during initiation of mtDNA replication ( Min et al Science adv 2021, Zhu et al. Cell 2022)
Aim 2. To establish the mechanisms of primer removal and ligation in the D-loop region
Work under this aim has been successful. We have demonstrated how mtDNA replication is terminated and how disturbed termination may lead to mtDNA defects. In three published papers (Posse et al., Nucleic Acids Res 2018; Matic et al., Nature Commun 2018; and Behadili et al., Nucleic Acids Res. 2018) we have elucidated the last step in mtDNA replication, when DNA synthesis is terminated and the 3´- and 5´-ends of the nascent strand are ligated together, to form a continuous circular mtDNA. Failure to ligate the mtDNA is causing a nick in the DNA, and our studies have identified the cellular activities required for proper ligation (MGME1, RNase H1, Ligase III, and the exonuclease activity of DNA polymerase gamma). We have also demonstrated how impaired function of these factors can cause linear deletion (e.g. MGME1, RNase H1 or partial duplications in mtDNA (MGME1, DNA polymerase gamma) ( Singh et al. NAR 2022, Misic et al. accepted NAR 2022)
Aim 3 To identify the mtDNA degradation machinery.
We have published one paper describing this line of work (Moretton et al. PLoS one 2017). A double-stranded break repair pathway similar to the one present in the nucleus does not exist in mitochondria. This observation has prompted us to investigate how mitochondria deal with double-stranded breaks. In our work we have targeted a restriction endonuclease to mitochondria and demonstrated that double-stranded breaks cause a rapid, and complete loss, of the damaged mtDNA molecule. None of the previously identified mitochondrial nucleases are involved in this process. In published work, we have established an RNAi platform and identified genes that affect mtDNA degradation. In ongoing work, we work to establish the molecular mechanisms underlying mtDNA degradation. It is worth mentioning that there have been some papers from other groups suggesting the possible model for degradation of linearized mtDNA molecules. Our findings so far do not support the published model, suggesting that there may be new, unexpected mechanisms waiting to be discovered.
Aim 4. To establish the mechanisms by which circular deletions are formed in mtDNA
The first phase of this project has been successfully completed. Somatic and germ line mtDNA deletions are associated with a variety of human diseases and biological ageing. However, the exact mechanisms of mtDNA deletion formation has not been well understood We have demonstrated that mtDNA deletions are formed via copy-choice recombination. To arrive at this conclusion, we used a combination of bioinformatics and in vitro biochemistry, in which we reconstitute deletion formation in the test tube (Persson et al, Nature Commun 2019). The work has opened up the possibility to develop drugs that can block deletion formation, which may be of importance for the treatment of different mitochondrial diseases and biological ageing. We are currently working on a couple of follow-up studies, to provide further support for our circular deletion formation model (Basu et al HMG 2020, Persson et al. manuscript 2022).
Aim 5. To establish a high-resolution structure of the mitochondrial replisome.
In this project, we have invested significant effort to establish the conditions required to form a stable mitochondrial replisome in vitro that can be subjected to structural analysis. The project has progressed steadily, but we have not yet arrived a molecular model of sufficient quality for publication. Even if the outcome of these type of structural studies are inherently difficult to predict, we do believe that we will be able to finish this very challenging aim within the time frame of the present ERC project. The project has however already generated a number of intermediate results that has been published, including a cryo-EM study of the mitochondrial DNA helicase TWINKLE, defining how disease-causing mutations affect the structure of this enzyme (Peter B et al., 2019 Hum Mol Genet, 2018). Furthermore, we are currently finalizing one manuscripts using cryo-EM to demonstrate how the LONP1-protease degrades components of the mitochondrial replisome (Shahzad et al., 2022 and Valenzuela et al. 2022).
