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

逆转座子是一类在大多数真核生物中普遍存在的基因组寄生虫，它们是这些基因组的主要组成部分。逆转录转座元件的复制会导致染色体的异常，包括插入、缺失和双链断裂，这可以影响细胞基因的表达，并作为新基因的来源。长长的散布元素(线条)在这一过程中发挥了核心作用。因此，由LINE编码的蛋白质正被探索为潜在的基因组工程的分子生物学工具。了解LINE蛋白如何与插入位点附近的DNA结合，对于了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。该项目将增强PI主要是本科机构的研究能力和研究计划，启动PI的职业生涯，并提供一台磷成像仪，供PI、研究助理以及其他STEM部门的研究人员和学生使用。为研究助理提供资金使低收入和多样化的学生能够获得研究。本科生将有机会参与生物化学和分子生物学的亲身实践原创研究，在会议上展示他们的研究成果，并发表研究结果。他们将发展他们的沟通和陈述技能，并将通过期刊俱乐部和PI和CO-PI之间每两周一次的联合实验室会议，为STEM领域的研究生学习和职业生涯做好更多准备。在这所研究密集型机构进行为期两周的带薪研究实习期间，与合作伙伴和研究生一起工作将进一步加强学生的研究培训，并提供建立网络的机会。在基因组突变和进化中起核心作用的线条整合机制与端粒延长和第二类内含子有相似的机制。该项目的重点是使用家蚕的R2Bm，这是一组早期的分支系，被称为限制性内切酶(RLE)系，以探索R2Bm蛋白如何与DNA结合，以及哪些DNA序列对结合R2Bm蛋白是重要的。位点特异性RLE品系因其特异性和编码单一的多功能基因产物而特别令人感兴趣。详细了解RLE株蛋白是如何与插入位点附近的DNA结合的，对于了解整个整合机制以及如何对蛋白质进行工程处理以改变位点特异性是必不可少的。此外，所提出的研究不仅将有助于了解模型元素R2Bm的整合机制，也将有助于类似元素的整合。该项目的目标是：(1)通过氨基酸替换来表征R2Bm蛋白质中一个重要的保守基序；(2)使用体外蛋白质DNA结合分析来分析蛋白质-DNA复合体；以及(3)由SELEX来表征DNA靶标要求。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.