Restricted Mobility of DNA Packaged in Phage PHI29 Viral Proheads Assessed by Single-Molecule Optical Tweezers Measurements of DNA Exit

Abstract

Many viruses contain DNA packed to such a high density that the mobility of the DNA inside the viral capsid is severely restricted, affecting the processes of DNA packaging and ejection. We study phage phi29, which uses an ATP-powered molecular motor to package DNA, by using optical tweezers to measure DNA ejection through the phi29 portal-motor channel after the removal of ATP during DNA packaging. We find that when initiated at low capsid filling levels, DNA exits faster than 10 kbp/s. When initiated at high filling levels, exit occurs with a dramatically reduced average velocity that decreases with increasing initial prohead filling. In individual exit measurements, complex dynamics and transient pausing are seen, which we attribute to the nonequilibrium DNA conformations thought to arise during DNA packaging. We also show that high concentration of Mg2+ slows exit dynamics, suggesting that the internal pressure of the confined DNA is the driving force for the ejection process.