BRC-BIO: Elucidating how protein-nucleic acid interactions create site-specific integration of retrotransposable elements

BRC-BIO：阐明蛋白质-核酸相互作用如何产生逆转录转座元件的位点特异性整合

• 批准号: 2217924
• 负责人: Varuni Jamburuthugoda
• 金额: $ 49.07万
• 依托单位: SUNY College at Geneseo
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2025-07-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2217924&HistoricalAwards=false
• 关键词: BRC; BIO; Elucidating; how; protein

Retrotransposable elements are a class of ubiquitous genomic parasites found in most eukaryotes, and they constitute a major component of these genomes. Replication of retrotransposable elements results in chromosomal aberrations, including insertions, deletions, and double stranded breaks, which can affect cellular gene expression and act as a source of new genes. Long INterspersed Elements (LINEs) have played a central role in this process. As such, the proteins encoded by LINEs are being explored as molecular biology tools for potential genome engineering. Knowledge of how the LINE protein binds to DNA near the insertion site is essential to understanding the integration mechanism and how the protein might be engineered to change site-specificity. The project will enhance the research capacity and research program at the PI’s primarily undergraduate institution, launch the PI’s career, and provide a phosphorimager, used by the PI, research assistants, and researchers and students from other STEM departments. Funding for research assistants makes research accessible to low-income and diverse students. Undergraduates will have opportunities to participate in hands-on original research in biochemistry and molecular biology, present their research at conferences, and publish the findings. They will develop their communication and presentation skills and will be more prepared for graduate studies and careers in STEM fields via journal clubs and bi-weekly joint lab meetings between the PI and CO-PI. Working with the Co-PI and graduate students during two-week paid research internships at the research-intensive institution will further student's research training and provides opportunities to network. The integration mechanism of LINEs, which plays a central role in genome mutation and evolution, shares mechanistic similarities to telomere elongation and group II introns. This project focuses on using R2Bm from Bombyx mori, an earlier branching group of LINEs known as Restriction Like Endonuclease (RLE) LINEs, to explore how the R2Bm protein binds to DNA and what DNA sequences are important for binding to the R2Bm protein. Site-specific RLE LINEs are of particular interest because of their specificity and because they encode a single multifunction gene product. A detailed knowledge of how the RLE LINE protein binds to DNA near the insertion site is essential to understanding the integration mechanism as a whole and how the protein might be engineered to change site-specificity. In addition, the proposed studies will shed light not only on the integration mechanism of the model element R2Bm but also on the integration of similar elements. The objectives of the project are: (1) to characterize an important conserved motif in the R2Bm protein by amino acid substitutions; (2) to analyze the protein-DNA complexes using in vitro protein DNA-binding assays; and (3) to characterize the DNA target requirements by SELEX.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.

逆转录元素是在大多数真核生物中发现的一类普遍存在的基因组寄生虫，它们构成了这些基因组的主要组成部分。逆转元素的复制导致染色体畸变，包括插入，缺失和双链断裂，这会影响细胞基因的表达并充当新基因的来源。长期散布的元素（线）在此过程中发挥了核心作用。因此，通过线条编码的蛋白质正在作为潜在基因组工程的分子生物学工具探索。了解线蛋白如何与插入位点附近的DNA结合的知识对于理解整合机制以及如何设计蛋白质以改变位点特异性至关重要。该项目将在PI的初级本科机构中增强研究能力和研究计划，启动PI的职业，并提供由PI，研究助理，研究人员以及其他STEM部门的研究人员和学生提供的磷光灯。研究助理的资金使低收入和多样化的学生可以进行研究。本科生将有机会参与动手生物化学和分子生物学的原始研究，在会议上介绍他们的研究并发表研究结果。他们将发展自己的沟通和演讲技巧，并通过期刊俱乐部和PI和Co-Pi之间的每两周一次的联合实验室会议为研究生学习和职业做好准备。在研究密集型机构为期两周的付费研究实习期间与Co-PI和研究生合作将进一步进一步的学生研究培训，并提供网络的机会。线的整合机制在基因组突变和进化中起着核心作用，与端粒伸长率和II组内含子具有机械相似性。该项目的重点是使用Bombyx Mori的R2BM，Bombyx Mori是一个较早的线条分支，称为限制性核酸内切酶（RLE）系，以探索R2BM蛋白如何与DNA结合，哪些DNA序列对于与R2BM蛋白结合至关重要。特定于位点的RLE线特别感兴趣，因为它们的特异性和编码单个多功能基因产物。关于RLE系蛋白如何与插入位点附近的DNA结合的详细知识，对于理解整个集成机制以及如何设计蛋白质以改变位点特异性至关重要。此外，拟议的研究不仅会阐明模型元素R2BM的整合机理，而且还阐明了相似元素的整合。该项目的目标是：（1）通过氨基酸取代表征R2BM蛋白中重要组成的基序； （2）使用体外蛋白DNA结合测定法分析蛋白质-DNA复合物； （3）以SELEX来表征DNA目标要求。该奖项反映了NSF的法定任务，并通过使用基金会的知识分子优点和更广泛的影响标准来评估，被认为是宝贵的支持。