Bulletin of the Korean Chemical Society

Korean Chemical Society (대한화학회)
권호 표지
DOI QR코드

DOI QR Code

Single-stranded DNA Enhances the Rate of Product Release During Nucleotide Hydrolysis Reaction by T7 DNA Helicase

  • Kim, Dong-Eun (Department of Biotechnology and Bioengineering, and Department of Biomaterial Control,Dong-Eui University) ;
  • Jeong, Yong-Joo (Department of Bio and Nanochemistry, Kookmin University)
  • Published : 2006.10.20
Download PDF
⟨ Previous Next ⟩

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 $Mg^{2+}$ which is necessary for the helicase-ssDNA complex formation. Steady state dTTP hydrolysis in the absence of $Mg^{2+}$ by the helicase-ssDNA complex provided $k_{cat}$ of $8.5\;{\times}\;10^{-3}\;sec^{-1}$. 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.

Keywords

References

  1. West, S. C. Cell 1996, 86, 177 https://doi.org/10.1016/S0092-8674(00)80088-4
  2. Patel, S. S.; Picha, K. M. Annu. Rev. Biochem. 2000, 69, 651 https://doi.org/10.1146/annurev.biochem.69.1.651
  3. Lohman, T. M.; Bjornson, K. P. Annu. Rev. Biochem. 1996, 65, 169 https://doi.org/10.1146/annurev.bi.65.070196.001125
  4. Ellis, N. A.; Groden, J.; Ye, T. Z.; Straughen, J.; Lennon, D. J.; Ciocci, S.; Proytcheva, M.; German, J. Cell 1995, 83, 655 https://doi.org/10.1016/0092-8674(95)90105-1
  5. Karow, J. K.; Chakraverty, R. K.; Hickson, I. D. J. Biol. Chem. 1997, 272, 30611 https://doi.org/10.1074/jbc.272.49.30611
  6. Yu, C. E.; Oshima, J.; Fu, Y. H.; Wijsman, E. M.; Hisama, F.; Alisch, R.; Matthews, S.; Nakura, J.; Miki, T.; Ouais, S.; Martin, G. M.; Mulligan, J.; Schellenberg, G. D. Science 1996, 272, 258 https://doi.org/10.1126/science.272.5259.258
  7. Shen, J. C.; Gray, M. D.; Oshima, J.; Loeb, L. A. Nucleic Acids Research 1998, 26, 2879 https://doi.org/10.1093/nar/26.12.2879
  8. Gray, M. D.; Shen, J. C.; Kamath-Loeb, A. S.; Blank, A.; Sopher, B. L.; Martin, G. M.; Oshima, J.; Loeb, L. A. Nature Genet. 1997, 17, 100 https://doi.org/10.1038/ng0997-100
  9. Eggleston, A. K.; West, S. C. Trends in Genetics 1996, 12, 20 https://doi.org/10.1016/0168-9525(96)81384-9
  10. Bujalowski, W.; Klonowska, M. M.; Jezewska, M. J. J. Biol. Chem. 1994, 269, 31350
  11. Patel, S. S.; Hingorani, M. M. J. Biol. Chem. 1993, 268, 10668
  12. Picha, K. M.; Patel, S. S. J. Biol. Chem. 1998, 273, 27315 https://doi.org/10.1074/jbc.273.42.27315
  13. Patel, S. S.; Rosenberg, A. H.; Studier, F. W.; Johnson, K. A. J. Biol. Chem. 1992, 267, 15013
  14. Hingorani, M. M.; Patel, S. S. Biochemistry 1993, 32, 12478 https://doi.org/10.1021/bi00097a028
  15. Ahnert, P.; Patel, S. S. J. Biol. Chem. 1997, 272, 32267 https://doi.org/10.1074/jbc.272.51.32267
  16. Hacker, K. J.; Johnson, K. A. Biochemistry 1997, 36, 14080 https://doi.org/10.1021/bi971644v
  17. Kim, D. E.; Patel, S. S. J. Mol. Biol. 2002, 321, 807 https://doi.org/10.1016/S0022-2836(02)00733-7
  18. Jeong, Y. J.; Levin, M. K.; Patel, S. S. Proc. Natl. Acad. Sci.USA 2004, 101, 7264 https://doi.org/10.1073/pnas.0400372101
  19. Hingorani, M. M.; Patel, S. S. Biochemistry 1996, 35, 2218 https://doi.org/10.1021/bi9521497
  20. Sambrook, J.; Fritsch, E. F.; Maniatis, T. In Molecular Cloning, A Laboratory Manual; 2nd ed.; Ford, N., Nolan, C., Ferguson, M., Eds.; Cold Spring Harbor Laboratorty Press: 1989
  21. Wong, I.; Lohman, T. M. Proc. Natl. Acad. Sci. USA 1993, 90, 5428 https://doi.org/10.1073/pnas.90.12.5428
  22. Ahnert, P.; Picha, K. M.; Patel, S. S. EMBO J. 2000, 19, 3418 https://doi.org/10.1093/emboj/19.13.3418
  23. Hingorani, M. M.; Washington, M. T.; Moore, K. C.; Patel, S. S. Proc. Natl. Acad. Sci.USA 1997, 94, 5012 https://doi.org/10.1073/pnas.94.10.5012

Cited by

  1. The Development of Miniaturized Extraction Tool for the Gene Biosensor vol.29, pp.1, 2006, https://doi.org/10.5012/bkcs.2008.29.1.269