Aim I: Identifying components of the mtDNA recombination machinery
Recent studies showed that repair mechanisms inside animal mitochondria are more diverse and effective than previously thought, but the existence of recombination-based repair remains controversial. My previous work showed that in Drosophila, double-strand breaks on one mitochondrial genotype could be repaired based on the homologous sequence of other co-existing genotypes, indicating that there is recombination-based repair machinery inside mitochondria. To identify proteins mediating such a repair, in the first three years, we performed a candidate screen and identified REC as a helicase that drives mtDNA recombination and repair (Klucnika et al., JCB 2022). During this period, another lab member focused on a genome-wide CRISPR screen to probe mtDNA damage response in human cells. So far, we have established RPE-Cas9-Tet3G cell lines expressing mitochondrially-targetted restriction enzymes upon Dox induction to cause DSBs in mitochondria. For the remaining period of this grant, we will transduce these cells with pooled sgRNA to identify proteins essential for cell survival upon mtDNA DSB damage.
Aim II: Developing a recombination toolkit to edit & map mtDNA
Probing interactions between mitochondrial genes has been challenging due to the multi-copy nature of mtDNA and the lack of genetic tools for editing and mapping mtDNA. Previously, my group established a system to isolate recombinant mitochondrial genomes in Drosophila. Using this toolkit, we found that sequence polymorphisms in one mitochondrial gene (mt:CoIII) could rescue the lethality caused by a detrimental mutation in another mitochondrial gene (mt:CoI). Through lipidomics profiling and biochemical and phenotypic analyses, we revealed that the two residues co-regulate cardiolipin binding to affect respiratory complex stability and activity. This work (Chiang et al, Nat Commun 2024) unwraps the complex intra-genomic interplays underlying mtDNA-linked disorders and how they influence disease expression.
Aim III: Establishing methods for the delivery of exogenous DNA into mitochondria
We tried multiple methods to deliver exogenous DNA into fly mitochondria. However, none of these approaches worked. We will continue to explore other methods proposed in the application. In the meantime, staff recruited to work on this part of the project investigated mtDNA maintenance during late spermatogenesis in parallel. This work identified POLDIP2 as an essential player in eliminating paternal mtDNA in late spermatids to guarantee the maternal inheritance of mitochondrial genomes (Wang*, Meerod*, Cortes-Silva*, Chiang* et al EMBO J, 2025).
