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

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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.