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

Biochemistry. 2000 Jan 11;39(1):205-12. doi: 10.1021/bi992105o.


Using a fluorescent sensor for inorganic phosphate, the kinetics of ATP hydrolysis by PcrA helicase were measured in the presence of saturating concentrations 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 the rapid phase is governed by the length of single-stranded DNA, while the slow phase is independent of its length. A kinetic model is presented in which the rapid phase is associated with translocation along single-stranded DNA, after the PcrA binds randomly along the DNA. There is a linear relationship between the length of single-stranded DNA and both the duration and amplitude of the rapid phase. These data suggest that the translocation activity occurs at 50 bases/s in unidirectional single-base steps, each requiring the hydrolysis of 1 ATP molecule.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphate / chemistry
  • Bacterial Proteins*
  • Base Composition
  • Base Sequence
  • Biological Transport
  • DNA Helicases / chemistry*
  • DNA Helicases / metabolism
  • DNA, Single-Stranded / chemistry*
  • DNA, Single-Stranded / metabolism
  • Geobacillus stearothermophilus / enzymology
  • Kinetics
  • Oligonucleotides / chemistry
  • Phosphates / chemistry
  • Poly T / chemistry
  • Subtilisins / chemistry*
  • Subtilisins / metabolism


  • Bacterial Proteins
  • DNA, Single-Stranded
  • Oligonucleotides
  • Phosphates
  • PrcA protein, bacteria
  • Poly T
  • Adenosine Triphosphate
  • Subtilisins
  • Adenosine Triphosphatases
  • DNA Helicases