| Protein complexes belonging to the super-family of ATPases Associated with diverse Activities of the Cell (AAA+), form rings that function as molecular motors to translocate DNA during important processes in the cell, including replication, recombination, transposition of genes, conjugation, genome segregation and viral DNA packaging.;We have carried out studies of the DNA packaging motor of bacteriophage Ф29, a motor with five ATPase subunits, directed at understanding the mechanism of force generation. Using optical-tweezers-based single molecule assays, we applied precise forces to these motors and followed the packaging dynamics.;First, we characterized the mechano-chemistry of the motor, how the chemical hydrolysis cycle of ATP couples to the mechanical cycle of pumping DNA. We studied the dependence of the motor's dynamics in response to a range of forces and chemical potentials. We have shown that the power stroke of the motor does not occur during the ATP binding, but during the release of phosphate. Next, by the use of non-hydrolysable ATP analogs, we showed that the firing of the motor-subunits is highly coordinated. Our findings allow us to postulate the first mechano-chemical scheme for this motor.;We then studied the nature of the interactions that allow the motor to bind and translocate DNA. Using synthetic DNA substrates that differed either in charge, chemical structure, or continuity, we probed the importance of various specific interactions. We found that the motor makes periodic contacts with the DNA backbone every 10 bp, tracking the 5'-3' strand but not the 3'-5' strand. The motor packaged actively against an applied force an un-charged substrate better than an abasic substrate. We also observe that the motor can translocate through large discontinuities or bulges in the DNA. We conclude that the motor likely holds and orients the DNA via ionic-interactions but sterically pushes on the DNA mostly via the bases using a non-specific 'friction drive'.;Our findings for the mechanism of force generation of the bacteriophage Ф29 DNA packaging motor constitute a significant advance in the understanding of the AAA+ DNA translocases. |
