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

植物线粒体双链断裂修复：产物和蛋白质

基本信息

批准号：
1933590
负责人：
Alan Christensen
金额：
$ 82万
依托单位：
University of Nebraska-Lincoln
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-15 至 2024-07-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1933590&HistoricalAwards=false
关键词：
Double strand break repair plant

项目摘要

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.

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的突变可以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线粒体DNA的修复和重排如何发生在植物拟南芥中。该项目将识别未知的（可能意想不到的）DNA修复酶，并将分析特定DNA断裂事件的后果。该项目将全年为高中生、本科生和研究生提供研究培训机会。一名高三学生也将通过青年内布拉斯加州科学家计划在实验室工作8周的暑期研究经验。该项目致力于从科学领域代表性不足的群体、潜在的第一代大学生或有经济需要的群体中招收学生。本项目将识别双链断裂（DSB）修复缺陷突变，并分析在体内特定位点诱导的DSB加工。筛选对环丙沙星敏感的突变体，可以在细胞器DNA中诱导DSB，从而确定细胞器DSB修复基因。三种限制性内切酶都能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钝端，5'-或3'-悬垂），它们将被一个诱导启动子驱动，并以线粒体为目标诱导断裂。在35个位点中，有些位点必须精确修复以维持功能，有些位点位于非编码、非保守区域或重复序列。修复结果将通过定量PCR、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。在突变背景中诱导断裂将有助于进一步了解DNA修复酶的功能，以及DSB修复途径之间的竞争。该奖项反映了美国国家科学基金会的法定使命，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。