Demonstration of unidirectional single-stranded DNA translocation by PcrA helicase: Measurement of step size and translocation speed

Using a fluorescent sensor for inorganic phosphate, the kinetics of ATP hyd rolysis by PcrA helicase were measured in the presence of saturating concen trations of oligonucleotides of various lengths. There is a rapid phase of inorganic phosphate release that is equivalent to several turnovers of the ATPase, followed by slower steady-state ATP hydrolysis. The magnitude of th e rapid phase is governed by the length of single-stranded DNA, while the s low phase is independent of its length. A kinetic model is presented in whi ch the rapid phase is associated with translocation along single-stranded D NA, after the PcrA binds randomly along the DNA. There is a linear relation ship between the length of single-stranded DNA and both the duration and am plitude of the rapid phase. These data suggest that the translocation activ ity occurs at 50 bases/s in unidirectional single-base steps, each requirin g the hydrolysis of 1 ATP molecule.