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Single Molecule Dynamics of Viral DNA Packaging Using Optical Tweezers

  • Author(s): Keller, Nicholas Andreas
  • Advisor(s): Smith, Douglas E
  • et al.
Abstract

Viral DNA packaging is a required step in the lytic cycle of many DNA viruses. A DNA polymer is packaged into a pre-formed capsid to a near crystalline density state by a powerful molecular motor that is fueled by ATP hydrolysis. To accomplish this task, the motor must generate enough force to match the resistance forces that arise from tight polymer confinement. These forces are due to entropy loss, bending energy, and electrostatic repulsion. Despite recent advances in the fields of viral DNA packaging and ejection, there are still many questions that remain unanswered. Does the packaged DNA undergo non-equilibrium dynamics? Do partly attractive DNA-DNA interactions as mediated by polyamines enhance or inhibit packaging? What affect does the conformational history of the packaged DNA have on the subsequent packaging dynamics? Which residues of the motor are responsible for generating force? And finally, is the force driving viral DNA ejection similar in magnitude to the package force? In this work, I present experimental studies that shed light on these questions. The packaging of single DNA molecules into single empty pro-capsids was measured using a custom-built dual optical tweezers system. Using the bacteriophage phi29 and T4 systems, I and my research collaborators pioneered new techniques in optical tweezers for investigating the dynamics of DNA packaging and force generation of a viral packaging motor. Using these techniques, we discovered many new findings in the areas of polymer confinement, non-equilibrium dynamics, DNA condensation, and enzyme kinetics.

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