Demonstration of unidirectional single-stranded DNA translocation by PcrA helicase: measurement of step size and translocation speed |
| |
Authors: | Dillingham M S Wigley D B Webb M R |
| |
Affiliation: | Sir William Dunn School of Pathology, University of Oxford, United Kingdom. |
| |
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. |
| |
Keywords: | |
本文献已被 PubMed 等数据库收录! |
|