The research component of this project focuses on the development and utilization of single molecule techniques to study the complex processes involved in DNA replication. The bacteriophage T7 DNA replication machinery will be examined. The mechanical manipulation of individual DNA molecules will be combined with the imaging of single fluorescently labeled proteins to study how double-stranded DNA is unwound and how this unwinding is coupled to DNA synthesis. By making 'molecular movies' of these processes at the single-molecule level, details and events can be observed that would otherwise be hidden under the ensemble average. The education component of this proposal strives to bridge the gap that exists between the physics education offered to biology graduate students and the increasing importance of quantitative thinking in biological research. A novel course will be developed that aims to train biology graduate and upper-level undergraduate students in the physical principles that are important in biological processes at the molecular scale. Interactive web-based, 'virtual' experiments will be created to let students develop an intuitive feeling for the role of physics in the behavior of biological macromolecules. The lecture materials will be converted into a textbook that is accompanied by a CD-ROM containing the interactive, virtual experiments.
The researchers involved in this project have developed new imaging techniques to visualize how DNA gets replicated. DNA replication is an important biological process that takes place when cells divide and its genome needs to be duplicated. Within a window as short as an hour, approximately 6 billion DNA basepairs need to be copied in a manner that leaves as few as possible mistakes. The cell has a number of dedicated proteins to perform this molecular tour de force. These proteins bind to the DNA, open it up, read it, and produce new DNA with the same content. Funded by the NSF, researchers at Harvard Medical School, led by Prof. Antoine van Oijen, have developed methods that allowed them to study how individual DNA molecules were replicated. They built optical microscopes and used sensitive CCD cameras to visualize a single DNA molecule and the proteins attached to it. By fluorescently tagging individual proteins with little molecular light bulbs, they we able to visualize where on the DNA these proteins were as the DNA replication reaction was progressing. By using these new tools, the researchers gained new understanding how these molecular Xerox machines perform their copying activities. In particular, they studied helicase, the protein that unzips the double-stranded DNA helix before the actual copying starts and the DNA polymerase, the protein that performs the actual copying. In a number of scientific publications, they describe how these single-molecule methods of dynamically visualizing such molecular processes open up new possibilities to study complicated biological processes at the fundamental, molecular level.
