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

M S Dillingham, D B Wigley, M R Webb

Research output: Contribution to journalArticle (Academic Journal)

187 Citations (Scopus)

Abstract

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.
Original languageEnglish
Pages (from-to)205-12
Number of pages8
JournalBiochemistry
Volume39
Issue number1
Publication statusPublished - 2000

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