Double-strand break repair in plant mitochondria: products and proteins

线粒体通常被称为“细胞的发动机”，因为它们产生细胞生命所需的能量。线粒体DNA的突变可能会导致动物患病和植物雄性不育，但线粒体如何修复受损的DNA却知之甚少。本研究的重点是了解线粒体DNA的修复和重排是如何在植物拟南芥中发生的。这个项目将识别未知的(可能是意想不到的)DNA修复酶，并将分析特定DNA断裂事件的后果。该项目将为高中、本科生和研究生提供全年的研究培训机会。一名高三学生还将获得资助，通过内布拉斯加州青年科学家计划，在实验室进行为期8周的暑期研究体验。该项目致力于招收来自科学界代表性不足的群体、潜在的第一代大学生或有经济需要的学生。该项目将识别双链断裂(DSB)修复缺陷的突变，并分析体内特定部位诱导的DSB的处理过程。筛选对环丙沙星敏感的突变体，将在细胞器DNA中诱导DSB，将识别细胞器DSB修复基因。三种限制性内切酶都识别线粒体基因组中相同的35个位置(但不同之处在于留下钝端、5‘-或3’-突出)，它们将由可诱导的启动子驱动，并定位于线粒体以诱导断裂。在这35个位点中，有些必须准确修复才能维持功能，有些位于非编码区、非保守区或重复序列。修复结果将使用定量聚合酶链式反应、熔融曲线分析和DNA测序进行分析。结果可能包括重排、突变或缺失。在突变背景中诱导中断将使人们能够进一步了解DNA修复酶的功能，以及DSB修复途径之间的竞争。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.