Elongin C complexes in Arabidopsis development and DNA repair

Elongin C 复合物在拟南芥发育和 DNA 修复中的作用

• 批准号: RGPIN-2014-04268
• 负责人: Schroeder, Dana
• 金额: $ 1.89万
• 依托单位: University of Manitoba
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=708143
• 关键词: Elongin; complexes; Arabidopsis; development; DNA

Plants depend on sunlight for survival, yet sunlight consists of not only beneficial visible light but also damaging ultraviolet (UV) rays. I am interested in how plants optimize their response to visible light while protecting themselves from UV damage. Here I propose to examine the role of Elongin C complexes in light-regulated developmental transitions, such as flowering, as well as in UV-damaged DNA repair. All organisms must protect their genomes (DNA) from UV damage. Amazingly, life-forms from yeast to plants to humans use basically the same process to do this: a pathway called Nucleotide Excision Repair, where UV-damaged DNA is recognized, unwound, cut and repaired. The only step in this process that varies between organisms is the initial damage recognition. In the baker's yeast Saccharomyces cerevisiae damage recognition and initiation of repair is performed by the Radiation Sensitive (RAD) 7 / RAD16 / Elongin C (ELC1) complex. In mammals (including humans) however, damaged DNA is recognized by Damaged DNA Binding protein (DDB) 1 & 2. In plants, work by my lab and other researchers has shown that DDB1&2 are involved in repair of plant UV-damaged DNA. Interestingly, recent bioinformatic analysis in the model plant Arabidopsis thaliana has revealed that plants also have RAD7, RAD16, and ELC1 proteins, but their functions are uncharacterized. This provides us with a unique opportunity to examine the roles and interactions of both the mammalian (DDB1&2) and yeast (RAD7&16) damaged DNA recognition factors in a single system. I propose to use the extensive molecular and genetic tools available to Arabidopsis researchers to examine the role of the RAD7/RAD16/ELC1 complex in plant UV tolerance and DNA repair. Specifically, we will generate genetic material with altered levels of these genes then analyze developmental, UV tolerance and DNA repair phenotypes. In addition, we will examine RAD7, RAD16 and ELC1 protein levels, interaction partners and location in the cell as well as regulation of these properties by UV. Excitingly, our preliminary results show that rad7a, rad7b, and rad16b mutants are all UV-sensitive, supporting our hypothesis and providing the first evidence in any multicellular organism of a role for the RAD7 complex in DNA repair. In addition, by analyzing development in our mutants, we have found that rad16 and elongin c mutants are early flowering. Thus the role of plant Elongin C complexes seems to extend beyond DNA repair to regulation of important growth parameters. In yeast and mammals Elongin C interacts not only with RAD7, but also with Elongin A. The Elongin A/C complex regulates transcript (RNA) elongation. We have identified a possible Arabidopsis Elongin A and are also interested in its function in plants and potential contribution to Elongin C's role in flowering time. Thus by examining the roles of these two prospective Elongin C complexes (RAD7- and Elongin A-containing) we will identify novel components of plant UV-damaged DNA repair as well as identify unique plant-specific roles for these conserved proteins, such as the regulation of flowering time. In this way we will gain insight into plant environmental response with respect to both stress (e.g. UV) and developmental (e.g. flowering time) cues. This research may lead to future crops with optimized UV tolerance and genome stability.

植物依靠阳光生存，但阳光不仅包括有益的可见光，还包括有害的紫外线（UV）。我感兴趣的是植物如何优化它们对可见光的反应，同时保护自己免受紫外线的伤害。在这里，我建议研究的作用，Elongin C复合物在光调节的发展转变，如开花，以及在紫外线损伤的DNA修复。 所有生物体都必须保护其基因组（DNA）免受紫外线损伤。令人惊讶的是，从酵母到植物再到人类的生命形式基本上都使用相同的过程来做到这一点：一种称为核苷酸切除修复的途径，在这种途径中，紫外线损伤的DNA被识别，解绕，切割和修复。在这个过程中，生物体之间唯一不同的步骤是最初的损伤识别。在面包酵母中，酿酒酵母损伤识别和修复的启动是由辐射敏感（RAD）7 /RAD 16/延伸蛋白C（ELC 1）复合物进行的。然而，在哺乳动物（包括人类）中，受损的DNA被受损的DNA结合蛋白（DDB）1和2识别。在植物中，我的实验室和其他研究人员的工作表明，DDB1&2参与修复植物紫外线损伤的DNA。有趣的是，最近在模式植物拟南芥中的生物信息学分析显示，植物也有RAD 7，RAD 16和ELC 1蛋白，但它们的功能是未知的。这为我们提供了一个独特的机会，在一个单一的系统中检查哺乳动物（DDB1&2）和酵母（RAD7&16）受损DNA识别因子的作用和相互作用。我建议使用广泛的分子和遗传工具，拟南芥研究人员研究的作用，RAD 7/RAD 16/ELC 1复合体在植物紫外线耐受性和DNA修复。具体来说，我们将产生这些基因水平改变的遗传物质，然后分析发育，紫外线耐受性和DNA修复表型。此外，我们还将研究RAD 7、RAD 16和ELC 1蛋白水平、相互作用伙伴和在细胞中的位置，以及紫外线对这些特性的调节。令人兴奋的是，我们的初步结果表明，rad 7a，rad 7 b和rad 16 b突变体都是紫外线敏感的，支持我们的假设，并提供了第一个证据，在任何多细胞生物体的作用，RAD 7复合物在DNA修复。 此外，通过对突变体的发育分析，我们发现rad 16和elongin c突变体是早花的。因此，植物延伸蛋白C复合物的作用似乎超越了DNA修复，以调节重要的生长参数。在酵母和哺乳动物中，延伸蛋白C不仅与RAD 7相互作用，而且与延伸蛋白A相互作用。延伸蛋白A/C复合物调节转录物（RNA）延伸。我们已经确定了一个可能的拟南芥Elongin A，也有兴趣在植物中的功能和Elongin C的作用在开花时间的潜在贡献。 因此，通过研究这两个潜在的延伸蛋白C复合物（含RAD 7和延伸蛋白A）的作用，我们将确定植物UV损伤DNA修复的新组分，并确定这些保守蛋白的独特植物特异性作用，如开花时间的调节。 通过这种方式，我们将深入了解植物对胁迫（如紫外线）和发育（如开花时间）线索的环境响应。这项研究可能会导致未来的作物具有优化的紫外线耐受性和基因组稳定性。