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

被引:188
作者
Dillingham, MS
Wigley, DB
Webb, MR
机构
[1] Univ Oxford, Sir William Dunn Sch Pathol, Oxford OX1 3RE, England
[2] Natl Inst Med Res, London NW7 1AA, England
关键词
D O I
10.1021/bi992105o
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:205 / 212
页数:8
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