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 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.
Dillingham
M S
MS
Sir William Dunn School of Pathology, University of Oxford, United Kingdom.
Wigley
D B
DB
Webb
M R
MR
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D001426:Bacterial Proteins; D001482:Base Composition; D001483:Base Sequence; D001692:Biological Transport; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D007700:Kinetics; D009841:Oligonucleotides / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D011071:Poly T / Q000737:chemistry; D013381:Subtilisins / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D010710:Phosphates; C102527:PrcA protein, bacteria; D011071:Poly T; D000255:Adenosine Triphosphate; D013381:Subtilisins; D000251:Adenosine Triphosphatases; D004265:DNA Helicases