Intellectual Merit: The project addresses a fundamental problem in biology: How do site- and structure-specific DNA-binding proteins locate their targets among a vast excess of nonspecific DNA? This question will be studied using a multi-disciplinary approach encompassing nanoscale engineering, surface chemistry, physics and biochemistry. The ability of proteins to locate rare targets is essential for all aspects of gene expression, DNA replication, chromosome dynamics and genome maintenance, yet meaningful details of how these processes occur are typically unavailable. This project will focus on post-replicative mismatch repair (MMR) as a model system, and experiments will be conducted to determine precisely how the MMR proteins MutSa and MutLa locate and respond to their specific DNA targets. Detailed mechanisms of these processes are not yet available, despite years of intensive investigation, largely due to the inherent limitations of ensemble-level biochemical measurements. To overcome these limitations, this project will utilize nanofabricated' DNA Curtains' and apply total internal reflection fluorescence microscopy (TIRFM) to directly visualize individual protein complexes as they search for and engage their target sites on individual molecules of DNA. This unique approach to single-molecule imaging was pioneered by the PI's laboratory, and enables rapid collection of statistically relevant information in real time from individual molecules by enabling parallel imaging of multiple reaction trajectories.
Broader Impacts - The technology developed in Dr. Greene's laboratory provides a 'high-throughput' approach for real-time, single-molecule analysis of nucleoprotein complexes. The PI has actively disseminated this technology to the broader research community, as it has the potential to provide novel insights into virtually any biological system that involves interactions between protein and DNA molecules. This interdisciplinary work also provides trainees with a cutting-edge, broad-based educational experience that will allow them to pursue significant scientific careers upon completion of their degree requirements. To promote the understanding of single-molecule approaches to biological science, these technologies have been integrated into the University's undergraduate and graduate course curriculum. The PI is engaged in concerted efforts to advance the scientific training and development of younger students, particularly through research-oriented educational programs. He serves as an undergraduate student mentor for Columbia University's Summer Undergraduate Research Fellowship program (SURF), and as a student mentor for the Harlem Children's Society. The project will integrate younger students into all aspects of scientific work performed in the laboratory, thereby providing them with valuable, real-world research experiences.
