Mechanism of a viral DNA packaging motor studied by characterization of biochemical mutants via optical tweezers measurements

Abstract

Optical tweezers measurements were employed to directly observe viral DNA packaging in wild type and packaging mutants of bacteriophage lambda. Several key findings are reported here: DNA packaging by purified wild type lambda motors was measured for the first time, showing nearly identical behavior in packaging DNA to crude extracts of terminase components. A slow packaging lambda mutant, T194M, was found to package DNA at ~10× slower velocity than wild type. Meanwhile another packaging mutant Y46F was found to package DNA slower than the wild type (60-70% the velocity of the wild type velocity) as well as slipping >10x more frequently (per length of DNA) than wild type. Another mutant (K84A) showed slower packaging (60-70% the velocity of wildtype), but displayed slipping and pausing behavior similar to wild type. Finally the pausing and slipping dependence on length of DNA packaged of the various terminases studied was discovered, suggesting further structural defects of the mutants that are detrimental to translocation. These studies confirm the location of an ATPase center in the N-terminal portion of gpA which is responsible for translocation of dsDNA.