EAGER: Repair and Recombination of Mitochondrial DNA

Brandeis University, Waltham MA

Investigators

Abstract

DNA damage, especially in the form of double-strand chromosome breaks (DSBs), poses a severe threat to genome integrity. Much has been learned about the various ways that eukaryotic cells can cope with DSB damage, through both homology-directed repair and nonhomologous end-joining processes, with one notable exception: virtually nothing is known about how DNA in mitochondria is repaired, in humans or even in the best-studied model organism, Saccharomyces cerevisiae. Understanding of how DSB repair and recombination occur in mitochondria may open the door to gene therapeutic approaches for the many human mitochondrial diseases that are related to defects in mtDNA. The project also provides research and career development opportunities for a post-doctoral scholar and undergraduate student. Budding yeast mitochondrial DNA (mtDNA) undergoes frequent homologous recombination when haploids conjugate and both parents contribute mtDNA to the zygote; but almost nothing is known about how these events are initiated or through what intermediates recombination progresses. In addition, yeast mitochondria exhibit very high levels of unidirectional genetic transfer (gene conversion) initiated by the site-specific endonuclease, I-SceI; but how such events occur is unknown, save for the fact that they are largely independent of the genes essential for analogous gene conversion processes occurring between chromosomes in the nucleus, including DSB repair events initiated by the same endonuclease. The goals of this EAGER project are threefold: 1) to use genome-wide screening of budding yeast by CRISPRi (inhibition of gene expression by conditional, dominant dCas9-mediated repression) to identify genes necessary for general homologous recombination between mtDNAs; 2) to identify specifically genes that are required for site-specific I-SceI endonuclease- mediated gene conversion; and 3) to seek mutants in yeast that would allow the uptake of DNA to effect gene editing within mitochondria. The last aim tackles a formidable challenge: to date it has not been possible to introduce DNA into the mitochondrion that could be used for gene editing (e.g. by Cas9-mediated template repair). This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.