Mitochondria are needed for converting food into usable energy forms (ATP) via the respiratory chain. Mitochondria have their own genomes (mtDNA). A typical somatic mammalian cell contains between 1,000 to 10,000 copies of mtDNA. The mtDNA encodes only for 13 essential...
Mitochondria are needed for converting food into usable energy forms (ATP) via the respiratory chain. Mitochondria have their own genomes (mtDNA). A typical somatic mammalian cell contains between 1,000 to 10,000 copies of mtDNA. The mtDNA encodes only for 13 essential proteins that are required for cellular ATP production. All proteins necessary for mtDNA replication, as well as transcription and translation of mtDNA genes, are encoded in the nucleus, translated in the cytoplasm and then imported in to the mitochondria. The mtDNA needs to be continuously copied in order to maintain correct mtDNA copy number to meet cellular energy demands. In the present project, we try to understand the molecular mechanisms of mtDNA replication in health and in disease. Defects in the mitochondrial replication machinery can lead to loss of genetic information by deletion and/or depletion of the mtDNA, which subsequently may cause disturbed energy production and neuromuscular symptoms in patients. An increased level of mutations in mtDNA is also an important factor in normal ageing. We are trying to understand the molecular mechanisms underlying these processes.
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. We are currently performing some final experiments and aim to submit a manuscript describing our findings during the fall of 2019.
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). This work is still ongoing and we plan to write yet another manuscript, now describing the role of RNase H1 in this process in the near future.
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 (Persso
We have established a new bioinformatics pipeline to detect mtDNA rearrangements using next generation sequencing of human cells (Persson et al. Nat Commun 2019). Our pipeline provides extensive benefits compared to long-range PCR, which has traditionally been used to detect mtDNA rearrangements. Our pipeline identifies mtDNA copy number changes, duplications, deletions, and point mutations in one single analysis. Using our pipeline, we have been able to identify three different types of mtDNA rearrangements (deletions, long duplications, and short duplications). We have also been able to identify and classify the proteins involved in the formation of these different rearrangements. Our work is currently being written up in a manuscript that we hope to publish in the near future (Basu et al., Manuscript 2019).
More info: https://falkenberglab.org/.