Summary

A large family of multimeric ATPases are involved in such diverse tasks as cell division, chromosome seg-regation, DNA recombination, strand separation, con-jugation, and viral genome packaging. One such sys-tem is the Bacillus subtilis phage f29 DNA packaging motor, which generates large forces to compact its genome into a small protein capsid. Here we use opti-cal tweezers to study, at the single-molecule level, the mechanism of force generation in this motor. We de-termine the kinetic parameters of the packaging mo-tor and their dependence on external load to show that DNA translocation does not occur during ATP binding but is likely triggered by phosphate release. We also show that the motor subunits act in a coordi-nated, successive fashion with high processivity. Fi-nally, we propose a minimal mechanochemical cycle of this DNA-translocating ATPase that rationalizes all of our findings