UC San Diego

The DNA packaging dynamics of bacteriophages [phi]29, [lambda], and T4 were studied at the single molecule level using a dual trap optical tweezers. Also, a method for producing long DNA molecules by PCR for optical tweezers studies of protein DNA interactions is presented and thoroughly characterized. This DNA preparation technique provided DNA samples for the [phi]29 and T4 studies. In the studies of [phi]29, the role of charge was investigated by varying the ionic conditions of the packaging buffer. Ionic conditions in which the DNA charge was highly screened due to divalent and trivalent cations showed the lowest resistance to packaging of the DNA to high density. This confirmed the importance of counterions in shielding the DNA interstrand repulsion when packaged to high density. While the ionic nature of the packaging buffer had a strong effect on packaging velocities, there was no clear trend between the counterion-screened charge of the DNA and the maximum packaging velocity. The packaging studies of [lambda] and T4 served as systems for comparative studies with [phi]29. Each system showed similarities to the [phi]29 system and unique differences. Both the [lambda] and T4 packaging motors were capable of generating forces in excess of 50 pN and showed remarkably high processivity, similar to [phi]29. However, dynamic structural transitions were observed with lambda that are not observed with [phi]29. The packaging of the [lambda] genome showed capsid expansion at approximately 30 percent of the genome packaged and capsid rupture at 90 percent of the genome packaged in the absence of capsid stabilizing protein gpD. Unique to the T4 packaging motor, packaging dynamics showed a remarkable amount of variability in velocities. This variability was seen both within individual packaging phages and from one phage to the next. This is possibly due to different conformational states of the packaging machinery. Additionally, [lambda] and T4 had average packaging velocities under minimal load of 600 bp/s and 700 bp/s, respectively, as compared to 140 bp/s for [phi]29