|
KMID : 0613820040140010131
|
|
Journal of Life Science
2004 Volume.14 No. 1 p.131 ~ p.140
|
|
|
Kinetic Measurement of the Step Size of DNA Unwinding by Bacteriophage T7 DNA Helicase gp4
|
|
Kim Dong-Eun
|
|
|
Abstract
|
|
|
|
T7 bacteriophage gp4 is the replicative DNA helicase that unwinds double-stranded DNA by utilizing dTTP hydrolysis energy. The quaternary structure of the active form of T7 helicase is a hexameric ring with a central channel. Single-stranded DNA passes through the central channel of the hexameric ring as the helicase translocates (5’→3’) along the single-stranded DNA. The DNA unwinding was measured by rapid kinetic methods and showed a lag before the single-stranded DNA started to accumulate exponentially. This behavior was analyzed by a kinetic stepping model for the unwinding process. The observed lag phase increased as predicted by the model with increasing double-stranded DNA length. Trap DNA added in the reaction had no effect on the amplitudes of double-stranded DNA unwound, indicating that the T7 helicase is a highly processive helicase. Global fitting of the kinetic data to the stepping model provided a kinetic step size of 10-11 bp/step with a rate of 3.7 s-1 per step. Both the mechanism of DNA unwinding and dTTP hydrolysis and the coupling between the two are unaffected by temperature from 4-37℃. Thus, the kinetic stepping for dsDNA unwinding is an inherent property of the replicative DNA helicase.
|
|
|
KEYWORD
|
|
|
DNA helicase, Hexameric ring, Rapid kinetics, Global fitting, Stepping model
|
|
|
FullTexts / Linksout information
|
|
|
|
|
|
Listed journal information
|
|
|