Mechanisms of Viral DNA Packaging

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DESCRIPTION (provided by applicant): In many viruses, an empty "prohead" is assembled and subsequently filled with DNA by the action of an ATP-dependent portal motor. DNA packaging occurs in many phages, herpesviruses, adenoviruses and poxviruses, and is one of the least understood steps in viral replication. In this remarkable process, the DNA s compacted to near-crystalline density--overcoming energetic penalties due to electrostatic repulsion, DNA bending stiffness, and entropy. A single molecule optical tweezers assay has enabled real time measurement of phage phi29 packaging and shows that the motor exerts forces of up to 60 pN on the DNA, making it one of the strongest molecular motors. DNA packaging in phi29 will be further studied using optical tweezers to determine the fundamental chemical and mechanical steps that generate motor movement, the biochemical kinetics of motor operation, and the nature of the motor-DNA interaction. Biochemical and structural studies suggest that the packaging machine may represent a new class of rotary motors. State-of-the- art single-molecule fluorescence and spectroscopy techniques will be developed to track conformational changes in the motor and test the rotary motor hypothesis. Application of new theoretical and mathematical modeling techniques will yield further insights. Results of biophysical measurements will be integrated with structure determination by X-ray crystallography and cryo-electron microscopy (from established collaborations) and used to guide the development of models. Single molecule studies of phage lambda DNA packaging will test the universality of packaging motors. Knowledge of the motor mechanism will aid the development of anti-viral drugs and yield insights into other DNA translocating enzymes such as polymerases helicases, recombination factors, and chromatin assembly factors. These studies will shed light on fundamental principles of protein-nucleic acid and protein-protein interactions, macromolecular self-assembly and conformational change, and mechanochemical energy transduction.