Mitochondria are often referred to as "the powerhouse of the cell" as they generate energy needed for cellular life. Mutations in mitochondrial DNA can cause disease in animals and male sterility in plants, but how mitochondria repair damaged DNA is poorly understood. The focus of this research is to understand how repair and rearrangement of mitochondrial DNA occurs in the plant, Arabidopsis thaliana. This project will identify unknown (and possibly unexpected) DNA repair enzymes, and will analyze the consequences of specific DNA breakage events. The project will provide research training opportunities for high school, undergraduate and graduate students throughout the year. A high school junior will also be sponsored to work in the lab for an 8-week summer research experience through the Young Nebraska Scientist program. This program strives to recruit students from groups that are underrepresented in science, potential first-generation college students, or those that have financial need.
This project will identify mutations defective in double-strand break (DSB) repair, and analyze processing of DSBs induced at specific sites in vivo. A screen for mutants hypersensitive to ciprofloxacin, which induces DSBs in organellar DNA, will identify organellar DSB repair genes. Three restriction enzymes, all of which recognize the same 35 sites in the mitochondrial genome (but which differ by leaving blunt ends, 5'-, or 3'- overhangs) will be driven by an inducible promoter and targeted to mitochondria to induce breaks. Of the 35 sites, some must be accurately repaired in order to maintain function, and some are in non-coding, non-conserved regions, or repeated sequences. The repair outcomes will be analyzed using quantitative PCR, melt-curve analysis and DNA sequencing. Outcomes could include rearrangements, mutations, or deletions. Induction of breaks in mutant backgrounds will enable further understanding of the functions of DNA repair enzymes, and the competition between pathways of DSB 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.
