RUI: Elucidating the mechanisms of site specific DNA cleavage using single molecule methods

Emmanuel College, Boston MA

Investigators

Abstract

This project will investigate how protein molecules are able to find specific locations in the genome accurately and quickly. Since there are over three billion base pairs in the human genome, this is akin to finding a needle in a haystack. Scientists have discovered that many proteins slide along the DNA during their search. Using highly sensitive techniques that can observe single molecules of DNA, this project will investigate how and why proteins slide. The proteins studied in this project, known as restriction endonucleases, cleave DNA into two strands once they bind to their target site. Many proteins that regulate and maintain the genome cleave DNA in their normal functioning. New techniques of genetic engineering also require cleavage of DNA at a specific site. This project will investigate how this process occurs. All laboratory work will be carried out by undergraduate students who will be trained in cutting edge technical skills as well as in scientific thinking and problem solving. Concepts from this research will be included in courses taught by the investigator. In addition, the investigator will hold workshops for students and teachers from Boston public schools. The workshops will expose high school students to scientific research and help teachers to create materials they can take back with them to use in their classrooms. Site specific DNA cleavage is a crucial step in antibody production, the prokaryotic adaptive immune system and genome editing. This project will investigate the target search strategies and cleavage mechanisms of a model system (restriction endonucleases) using a combination of single-molecule techniques and computational modeling. In one technique, microbeads tethered with DNA will be used to measure the exact time of cleavage of individual DNA molecules. In a single experiment, hundreds of DNA can be measured to yield high resolution kinetic data. A second technique will use fluorescence to track the diffusion of individual proteins along DNA. One dimensional diffusion constants, as well as off-rates, can be determined from single particle trajectories. Theoretical models of target search will be developed and compared to experimental data. Computer simulations of random walks, as well as models based on chemical kinetics, will be explored. The PI will train undergraduate students in research, scientific communication and scientific writing. Modules on tethered Brownian motion and DNA cleavage will be introduced into a biochemistry course. In outreach activities, half day research experiences for Boston area high school students will be held. In addition, a professional development course for high school teachers will be created.