UC San Diego

DNA packaging is a major step in the process of assembly of many viruses, including many dsDNA bacteriophages (bacterial viruses). An ATP-powered molecular motor translocates a dsDNA molecule into the viral prohead shell where it is confined to near-crystalline density. Our studies concern efforts to understand how this multi-subunit nanoscale motor functions to transfer ATP chemical energy to mechanical work as needed to translocate DNA against resisting load forces. In this dissertation, optical tweezers, a method for real-time, high force, and small displacement measurements on single DNA molecules, was employed to conduct a series of experiments on bacteriophage T4, φ29 and Lambda DNA packaging mechanisms. Improved instrument calibration methods and novel data analysis methods were also introduced. We used measurements of DNA elasticity in the linear, high-force regime to determine trap compliance more-reliably, simultaneously with other calibration parameters. New methods for pause and slip detection, data segmentation, and motor velocity determination were also explored. A major focus was investigation into whether substrate DNA sequence affects motor function. Evidence against a ‘B-A Scrunchworm’ model was obtained by showing T4 translocation dynamics are insensitive to A-philic DNA sequences. Preliminary data was also obtained supporting the same result for the φ29 motor. For T4, which exhibits large motor velocity fluctuations and pausing/slipping, more general evidence against sequence-dependent motor function was obtained from analyses looking for correlations across many events recorded with the same or different sequences. Phage lambda packaging was measured with improved resolution and with translocation interrupted by a putative ADP release inhibitor (Na3VO4) previously found with φ29 to cause isolated bursts of translocation steps ~10 bp in size. For lambda, preliminary evidence was obtained suggesting a different quantized translocation size of ~5 bp and a distinct packaging-slip-pause behavior. Lastly, preliminary studies of the T4 motor found no significant slowing with a high concentration of added phosphate, suggesting that phosphate release following ATP hydrolysis is essentially irreversible, consistent with prior reported findings on φ29 packaging. Preliminary T4 measurements with Na3VO4 revealed significantly slowed packaging but quantized translocation steps could not be detected because all translocation events were interrupted by variable size slips.