Intellectual Merit: For many large DNA viruses, the production of new viruses requires use of a protein molecular motor, powered by chemical energy from ATP, to force the replicated viral DNA into the preformed empty viral shell, which is also made of protein. The viral DNA is replicated as a continuous long chain that must be cut into complete, unit-length pieces to provide each new viral particle with a complete DNA genome. The protein motor that packages the DNA also cuts the DNA chain to the correct length. For the herpes viruses and many bacterial viruses, the DNA is cut at specific sites to generate viral DNA having correct ends. Packaging begins with a precise initial cut, which generates the DNA end that is threaded into the shell by the motor. Once a complete viral DNA has been packaged, the motor nuclease again precisely cuts the DNA to terminate the DNA packaging cycle. This project aims to determine the mechanism by which the terminal DNA cut is precisely made. The model virus to be used in these studies is called "lambda," and it infects the bacterium E. coli. It was chosen because it is one of the most favorable systems for investigating this mechanism. Lambda has a special recognition site, adjacent to the proper cut site, to stop the packaging motor, so that the terminating cut is precisely made. The efficiency of termination increases with the length of the packaged DNA. As the shell fills, the resistance to packaging more DNA increases, the velocity of packaging slows, and more energy is required for the motor to continue packaging. Models to explain how termination efficiency is controlled by the extent of packaging emphasize (1) the extent of shell filling, (2) the packaging energy required, or (3) that packaging velocity regulates termination. In this project, each of these factors will be studied to determine which controls the termination process. These studies will be facilitated by the use of "optical tweezers," a powerful, biophysical technique in which single DNA molecules and individual virus shells are attached to plastic microspheres and manipulated with focused laser beams. Packaging of a single DNA molecule can be measured by detecting the movement of the microspheres and the forces acting on them. The rate of DNA movement, the energy required for packaging, and the extent of packaging will be manipulated through genetic, biochemical, and biophysical perturbations. The roles of a putative shell-stabilizing protein and of various regions of the motor proteins will also be explored using genetic and biochemical methods. The project will increase understanding of how viruses move, cut and package DNA.
Broader Impacts: Viral DNA packaging motors not only play an important role in the assembly of many viruses, but very likely also share structural features and mechanisms with many other cellular, DNA-processing enzymes, including helicases, bacterial chromosome segregation motors, and endonucleases. A strong interdisciplinary educational environment, integrating concepts and methods from the biological and physical sciences, will be ensured for participating graduate and undergraduate students. The project is led by two experienced researchers with complementary research backgrounds in biophysics and molecular biology, and strong records of research mentoring of undergraduates and students from diverse backgrounds. New undergraduate biophysics course materials will also be developed in the course of the project. Educational materials for K-12 science education will be developed in collaboration with the R.H. Fleet Science Center.
This project is jointly funded by the Genetic Mechanisms cluster in the Division of Molecular and Cellular Biosciences and by the Chemistry of Life Processes program in the Division of Chemistry.
