University of Cambridge
Safeguarding the mitochondrial genome: identifying components of the mtDNA repair machinery
Abstract
dc:description.abstractMechanisms that safeguard mitochondrial DNA (mtDNA) are crucial for limiting the accumulation of mutations associated with mitochondrial dysfunction and age-related diseases. Despite this, the pathways responsible for repairing double-strand breaks (DSBs) in animal mitochondria remain poorly characterised. This thesis explores mtDNA repair mechanisms across diverse species, including Drosophila melanogaster, humans, and corals, aiming to uncover the molecular processes underlying mitochondrial genome maintenance. The project initially utilised a candidate-driven approach, focusing on REC and its interacting partners. REC, the Drosophila ortholog of human MCM8, is an MCM helicase that is known to be involved in meiotic recombination. However, recent work in our lab showed that it also localises to mitochondria in Drosophila and human cells, suggesting that nuclear DNA repair proteins could be co-opted to maintain mitochondrial genome integrity. I performed phenotypic assays and showed that compromising REC function affects organismal fitness during ageing. Additionally, I tagged interacting partners of REC, as well as other proteins involved in meiotic recombination, including MRE11, NBS, SPN-A, SPN-B, SPN-D, Okra, MEI-9, MEI-217, MEI-218, and HDM, to investigate their subcellular distribution in Drosophila. Among these proteins, Okra, SPN-B and MEI-217 exhibited potential mitochondrial enrichment. The candidate-focused approach is limited in its ability to screen for large numbers of novel mtDNA repair proteins. Consequently, the second phase of my project employed two systematic strategies to characterise mtDNA damage repair. The first approach involved RNA-seq to profile transcriptional responses to mtDNA damage. Specifically, DSBs in the mtDNA of Drosophila somatic tissues and human HEK293T cells were induced via the expression of mitochondrially targeted restriction enzymes. While this approach did not identify strong candidates, it led to the development of robust tools for inducing mtDNA damage in vivo. The second strategy employed DeepLoc 2.0, a deep learning-based algorithm, to predict DNA repair proteins with potential mitochondrial localisation in Drosophila and humans. I then attempted to validate 17 of the predicted candidates via overexpression experiments in cell lines. I further tested 5 of the most promising candidates via endogenous tagging in Drosophila but found no clear mitochondrial enrichment of these proteins. However, additional experiments in cell lines indicate that two of these proteins may localise to mitochondria in human cells. In parallel, I collaborated with a research group in Okinawa to investigate mtDNA maintenance in corals, which exhibit some of the lowest mtDNA mutation rates among animals. Analysis of assembled mitochondrial genomes from Palythoa corals revealed remarkably low single nucleotide polymorphism (SNP) levels across geographically distant populations. This suggests enhanced mtDNA replication and repair mechanisms. Using DeepLoc 2.0, two DNA repair proteins were identified in a group of corals called hexacorals but were absent in other cnidarian species with higher mutation rates. These findings point to unique mtDNA maintenance strategies in hexacorals. To validate these computational predictions, novel subcellular proteomics approaches were developed for use in cnidarians. Together, these studies provide new insights into the diversity of mtDNA repair mechanisms in animals and uncover potential species-specific adaptations for mitochondrial genome maintenance.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2025
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- McCormack, Matthew
- Advisors dc:contributor.advisor
-
- O'Kane, Cahir
- Ma, Hansong
Subjects
dc:subject × 13Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.117734
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/383238