CAREER: Using High-throughput Single-molecule Analysis to Reveal the Mechanisms of Target Site Location by DNA Repair Proteins

This research incorporates micro-scale materials engineering, surface chemistry, physics and biochemistry to answer fundamental questions about biology that cannot easily be addressed through traditional experimental methods. The overall goal encompasses a classic problem in biochemistry, namely: How do site- or structure-specific DNA-binding proteins locate their targets among a vast excess of nonspecific DNA? To help address this question, the Greene laboratory is using total internal reflection fluorescence microscopy (TIRFM) as a tool to directly visualize individual protein complexes as they search for their target sites on single molecules of DNA. The Greene laboratory is also developing new methods that will allow the construction of aligned arrays comprised of hundreds of individual DNA molecules, which are suspended above an inert lipid bilayer and organized into patterns with user-defined orientations, tensions, and topologies. These DNA arrays will allow for rapid collection of statistically relevant information from hundreds of individual molecules by making possible parallel processing of multiple reaction trajectories. These novel research tools will be used to determine how proteins that are involved in post-replicative repair of mismatched bases locate and respond to their specific targets. Despite years of intensive investigation these mechanisms remain unknown, largely due to the inherent limitations of traditional ensemble-level biochemical measurements. These new single-molecule approaches can be used to determine exactly what proteins are bound to DNA, where they are bound, how they behave, when they leave, and how they influence one another, all in real-time at the single-molecule level. The technology-driven methods developed during the course of this research will provide a high-throughput approach for single-molecule analysis of nucleoprotein complexes, which can be applied towards the study of virtually any biological system that involves the 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 successfully contribute to the scientific community upon completion of their degree requirements. To promote the understanding of single-molecule approaches, these emerging technologies will be integrated into the university's graduate course curriculum; several departmental lectures will be scheduled featuring leading experts from around the country; and a regional discussion group/symposium will also be organized to stimulate interactions and communication between the laboratories in the New York area that are interested in single-molecule research. Dr. Greene has initiated a separate project that will be conducted solely by undergraduate and high school students. The goal of these efforts is to incorporate younger students into all aspects of scientific work performed in the laboratory, thereby providing them with valuable, real-world research experience.