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

逆转录转座因子是一类普遍存在于真核生物中的基因组寄生物，是真核生物基因组的主要组成部分。逆转录转座因子的复制导致染色体畸变，包括插入、缺失和双链断裂，其可影响细胞基因表达并作为新基因的来源。长间隔元素（LINE）在这一过程中发挥了核心作用。因此，LINE编码的蛋白质正在被探索作为潜在基因组工程的分子生物学工具。LINE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蛋白质如何被工程化以改变位点特异性至关重要。该项目将提高PI主要本科机构的研究能力和研究计划，启动PI的职业生涯，并提供PI，研究助理以及其他STEM部门的研究人员和学生使用的磷光成像仪。为研究助理提供资金，使低收入和多样化的学生能够进行研究。本科生将有机会参与生物化学和分子生物学的动手原创研究，在会议上展示他们的研究，并发表研究结果。他们将发展他们的沟通和演讲技巧，并将通过期刊俱乐部和PI和CO-PI之间的双周联合实验室会议为STEM领域的研究生学习和职业生涯做好更多准备。在研究密集型机构为期两周的带薪研究实习期间与Co-PI和研究生合作，将进一步促进学生的研究培训，并提供网络机会。LINEs的整合机制在基因组突变和进化中起着核心作用，与端粒延伸和II组内含子具有相似的机制。该项目的重点是使用来自家蚕的R2 Bm，这是一种早期的LINES分支，称为限制性内切酶（RLE）LINES，以探索R2 Bm蛋白如何与DNA结合以及哪些DNA序列对R2 Bm蛋白的结合很重要。位点特异性RLE LINE是特别感兴趣的，因为它们的特异性，因为它们编码一个单一的多功能基因产物。详细了解RLE LINE蛋白如何与插入位点附近的DNA结合，对于理解整个整合机制以及如何改造蛋白质以改变位点特异性至关重要。此外，所提出的研究将揭示不仅在集成机制的模式元素R2 Bm，但类似的元素的集成。本研究的主要目的是：（1）通过氨基酸替换来表征R2 Bm蛋白中的一个重要保守基序：（2）利用体外蛋白质DNA结合分析来分析蛋白质-DNA复合物;以及（3）该奖项反映了NSF的法定使命，并被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准。

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.