Pathogenic mechanisms affecting mitochondrial DNA replication and transcription
Abstract
Mitochondria are cytoplasmic organelles fundamental to life and health. In mitochondria, energy from the food we eat is converted into adenosine triphosphate (ATP), which in turn is used as a source of chemical energy to drive a multitude of cellular reactions. Owing to its endosymbiotic origin, the mitochondrion contains its own genetic material, a circular double-stranded DNA molecule (mtDNA) of about 16.6 kb. The mitochondrial genome contains 37 genes, which code for 13 protein components of the oxidative phosphorylation system, 2 ribosomal RNAs that are required for mitochondrial ribosome biogenesis, and a set of 22 transfer RNAs.
The enzymatic systems needed to replicate and transcribe mtDNA are distinct from those present in the nucleus. The objective of this thesis is to characterize pathogenic mechanisms affecting mtDNA replication and transcription in human cells. In collaboration with clinical colleagues, we report that mutations in the gene coding for mitochondrial RNA polymerase (POLRMT) can cause mitochondrial dysfunction and neurological disease. Using in vitro biochemistry and cell biology approaches, we find that the identified mutations cause deleterious effects on mitochondrial transcription, which in turn impair biogenesis of the oxidative phosphorylation system.
Mammalian mitochondria lack systems for ribonucleotide excision repair and mtDNA therefore contains relatively high levels of embedded ribonucleotides, which can be even higher in patients suffering from genetic disorders associated with imbalanced nucleotide pools. We demonstrate that embedded ribonucleotides can cause problems for the transcribing POLRMT, causing premature termination of mitochondrial transcription. We suggest that these effects can contribute to the phenotypes associated with nucleotide pool disorders.
Others have demonstrated that nucleoside analogues used to treat retroviral infections can cause a progressive accumulation of somatic mtDNA mutations. We investigated the effects of two nucleoside analogues commonly used to treat childhood cancer, 5-Fluorouracil (5-FU) and 6-Thioguanine (6-TG). Using a reconstituted mtDNA replication system, with highly purified components, we demonstrate that both 5-FU and 6-TG impair the activity of mitochondrial DNA polymerase in vitro. We also find that these compounds can cause mtDNA replication stalling in cells. Taken together, our data suggest that 5-FU and 6-TG have the potential to cause mutations, but future studies of mtDNA isolated from patients treated with these compounds are required to validate this idea.
Parts of work
1. Ribonucleotides embedded in template DNA impair mitochondrial RNA polymerase progression.
Meenakshi Singh, Viktor Posse, Bradley Peter, Maria Falkenberg and Claes M. Gustafsson. Nucleic Acids Research, 2022, Vol. 50, No. 2, 989–999. https://doi.org/10.1093/nar/gkab1251
https://doi.org/10.1093/nar/gkab1251 2. POLRMT mutations impair mitochondrial transcription causing neurological disease.
Monika Oláhová, Bradley Peter, Zsolt Szilagyi, Hector Diaz-Maldonado, Meenakshi Singh, Ewen W. Sommerville, Emma L. Blakely, Jack J. Collier, Emily Hoberg, Viktor Stránecký, Hana Hartmannová, Anthony J. Bleyer, Kim L. McBride, Sasigarn A. Bowden, Zuzana Korandová, Alena Pecinová, Hans-Hilger Ropers, Kimia Kahrizi, Hossein Najmabadi, Mark A. Tarnopolsky, Lauren I. Brady, K. Nicole Weaver, Carlos E. Prada, Katrin Õunap, Monica H. Wojcik, Sander Pajusalu, Safoora B. Syeda, Lynn Pais, Elicia A. Estrella, Christine C. Bruels, Louis M. Kunkel, Peter B. Kang, Penelope E. Bonnen, Tomáš Mráček, Stanislav Kmoch, Gráinne S. Gorman, Maria Falkenberg, Claes M. Gustafsson and Robert W. Taylor. Nature Communications, 2021, Vol. 12, No. 1, 1135.
https://doi.org/10.1038/s41467-021-21279-0 3. 5-Fluorouracil and 6-Thioguanine impair mitochondrial DNA replication.
Meenakshi Singh, Louise Jenninger, Emily Hoberg, Laleh Arabinian, Xuefeng Zhu, Claes M. Gustafsson and Maria Falkenberg.
Manuscript to be submitted.
Degree
Doctor of Philosophy (Medicine)
University
University of Gothenburg. Sahlgrenska Academy
Institution
Institute of Biomedicine. Department of Medical Biochemistry and Cell Biology
Disputation
Torsdagen den 25 maj 2023, kl. 13.00, Hörsal Arvid Carlsson, Academicum, Medicinaregatan 3, Göteborg
https://gu-se.zoom.us/j/68730572178?pwd=ejJVdGIwbHhoTzVxN0tvQmJQOXVxUT09
Date of defence
2023-05-25
meenakshi.singh@gu.se
meenakshisingh@live.com
Date
2023-04-24Author
Singh, Meenakshi
Keywords
mtDNA
ribonucleotides
nucleoside analogues
POLRMT
Publication type
Doctoral thesis
ISBN
ISBN 978-91-8069-235-9 (PRINT)
ISBN 978-91-8069-236-6 (PDF)
Language
eng