Homologous recombination and its application in manipulating animal mitochondrial dna
Mitochondrial DNA (mtDNA) is a multi-copy genome that works with the nuclear genome to control energy production and various cellular processes. To date, disorders associated with mutations in mtDNA are among the most common genetically inherited metabolic diseases1. However, our knowledge regarding many aspects of mtDNA biology remains limited, and we know even less about how it influences development and organismal traits. This is largely due to our inability to manipulate mtDNA. Recently, a colleague and I developed novel genetic tools in Drosophila that allowed us to isolate animal mitochondrial mutants for the first time, and to create heteroplasmic organisms containing two mitochondrial genotypes2,3. These advances make Drosophila a powerful system for mtDNA studies. Importantly, I showed that Drosophila mtDNA could undergo homologous recombination. Furthermore, I established a system to induce recombination at specific sites and select for progeny containing only the recombinant genome4. Thus, my work has demonstrated the existence of recombination in animal mitochondria, and opens up the possibility of developing a recombination system for functional mapping and manipulating animal mtDNA. Here I propose to 1) identify components of the mitochondrial recombination machinery by a candidate RNAi screen; 2) develop a recombination toolkit to map trait-associated mtDNA sequences/SNPs; and 3) build a site-directed mutagenesis system by establishing robust ways to deliver DNA into fly mitochondria. Given the essential functions of mitochondria and their involvement in incurable diseases, the genetic tools developed in this proposal will transform the field by making it possible to link mtDNA variations to phenotypic differences and introduce specific mutations into mtDNA for functional studies at organismal level. These advances will open many possibilities to accelerate our understanding on how mtDNA impacts health, disease and evolution.