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

线粒体通常被称为“细胞的发电站”，因为它们产生细胞生命所需的能量。 线粒体DNA的突变会导致动物疾病和植物雄性不育，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植物拟南芥中线粒体DNA的修复和重排是如何发生的。该项目将确定未知的（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。该项目将为高中生、本科生和研究生提供全年的研究培训机会。一名高中三年级学生也将被赞助在实验室工作，通过内布拉斯加州青年科学家计划进行为期8周的夏季研究体验。本项目面向科学领域的弱势群体、第一代大学生、有经济困难的学生等进行招生。本项目将鉴定双链断裂（DSB）修复的缺陷突变，并分析在体内特定部位诱导的DSB的加工过程。筛选对环丙沙星敏感的突变体将鉴定细胞器DSB修复基因，环丙沙星诱导细胞器DNA中的DSB。三种限制性内切酶都识别线粒体基因组中相同的35个位点（但不同之处在于留下平端、5 '-或3'-突出端），它们将由诱导型启动子驱动并靶向线粒体以诱导断裂。在这35个位点中，有些必须被精确修复以维持功能，有些则位于非编码区、非保守区或重复序列中。将使用定量PCR、熔解曲线分析和DNA测序来分析修复结果。结果可能包括重排、突变或缺失。突变体背景中断裂的诱导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知识价值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.