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Bulletin of the Korean Chemical Society (BKCS)

ISSN 0253-2964(Print)
ISSN 1229-5949(Online)
Volume 27, Number 10
BKCSDE 27(10)
October 20, 2006 

 
Title
Single-stranded DNA Enhances the Rate of Product Release During Nucleotide Hydrolysis Reaction by T7 DNA Helicase
Author
Dong-Eun Kim, Yong-Joo Jeong*
Keywords
DNA helicase, M13 single-stranded DNA, Pre-steady state kinetics, Chemical quench-flow technique
Abstract
Bacteriophage T7 gp4A' is a ring-shaped hexameric DNA helicase that catalyzes duplex DNA unwinding using dTTP hydrolysis as an energy source. To investigate the effect of single-stranded DNA (ssDNA) on the kinetic pathway of dTTP hydrolysis by the T7 DNA helicase complexed with ssDNA, we have first determined optimal concentration of long circular M13 single-stranded DNA and pre-incubation time in the absence of Mg2+, which is necessary for the helicase-ssDNA complex formation. Steady state dTTP hydrolysis in the absence of Mg2+ by the helicase-ssDNA complex provided kcat of 8.5× 10-3 sec-1. Pre-steady state kinetics of the dTTP hydrolysis by the pre-assembled hexameric helicase was monitored by using the rapid chemical quench-flow technique both in the presence and absence of M13 ssDNA. Pre-steady state dTTP hydrolysis showed distinct burst kinetics in both cases, indicating that product release step is slower than dTTP hydrolysis step. Pre-steady state burst rates were similar both in the presence and absence of ssDNA, while steady state dTTP hydrolysis rate in the presence of ssDNA was much faster than in the absence of ssDNA. These results suggest that single-stranded DNA stimulates dTTP hydrolysis reaction by T7 helicase by enhancing the rate of product release step.
Page
1618 - 1622
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