How to Mend a Broken Mitochondrial Genome: Mitochondrial Recombination And Its Applications

Animal mitochondria produce cellular energy and rely on genes encoded within their own genome (mtDNA) in addition to those in the nucleus. mtDNA mutations are linked to mitochondrial and age-related diseases. Therefore, safeguarding mechanisms are crucial to limit mtDNA mutations and curtail ageing and disease. Yet, pathways that repair animal mtDNA double-strand breaks remain uncharacterised. Double-strand breaks can be accurately repaired by homologous recombination but spontaneous mtDNA recombination is only detected rarely in animals. A system to induce and select for mtDNA recombination was recently developed in the fruit fly, Drosophila melanogaster. In this thesis, I used this system to investigate the mechanism of animal mtDNA recombination and its applications for mitochondrial genetics. First, I identified candidates that may mediate mtDNA recombination by performing a screen for nuclear repair proteins that are enriched in mitochondria when overexpressed in Drosophila cell culture. This identified mitochondrial localisation of REC, a helicase that drives meiotic crossovers in the nucleus. I then performed mtDNA recombination assays and found that REC is required for mtDNA recombination in Drosophila germline and somatic tissues. Moreover, loss of REC increased age-induced mitochondrial mutation load and dysfunction, showing that REC safeguards mtDNA during ageing. Next, I investigated whether the mitochondrial function of REC is conserved in humans. The human homologue of REC, MCM8, and its interacting partner, MCM9, localise to mitochondria and human cells with mutated MCM8 accumulate more mtDNA mutations. Therefore, MCM8 also functions to safeguard mtDNA. To gain further insight into the mechanism of Drosophila mtDNA recombination, I examined whether other nuclear recombination factors localise to mitochondria in vivo. Nearly all factors examined were not enriched in mitochondria, suggesting that mitochondrial recombination may occur by a different mechanism to that in the nucleus. Finally, I utilised the system to induce mtDNA recombination to develop forward and reverse genetic tools to study mtDNA. Techniques to edit animal mtDNA are desperately needed to better understand the consequences of mtDNA mutations on disease and ageing.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2021

Author dc:creator

• Klucnika, Anna

Advisor dc:contributor.advisor

• Ma, Hansong

• Mitochondria
• Genetics
• DNA repair
• Recombination

Licence

• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.86278

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/338873