Helicases pp 57-83 | Cite as

Experimental and Computational Analysis of DNA Unwinding and Polymerization Kinetics

  • Manjula Pandey
  • Mikhail K. Levin
  • Smita S. Patel
Part of the Methods in Molecular Biology book series (MIMB, volume 587)


DNA unwinding and polymerization are complex processes involving many intermediate species in the reactions. Our understanding of these processes is limited because the rates of the reactions or the existence of intermediate species is not apparent without specially designed experimental techniques and data analysis procedures. In this chapter we describe how pre-steady state and single-turnover measurements analyzed by model-based methods can be used for estimating the elementary rate constants. Using the hexameric helicase and the DNA polymerase from bacteriophage T7 as model systems, we provide stepwise procedures for measuring the kinetics of the reactions they catalyze based on radioactivity and fluorescence. We also describe analysis of the experimental measurements using publicly available models and software gfit (http://gfit.sf.net).

Key words

Hexameric helicase replication DNA unwinding T7 bacteriophage DNA polymerase DNA synthesis strand displacement primer extension gfit global regression analysis 



We thank the Patel lab members for proofreading the chapter and testing the models. This work was supported by National Institute of Health grant (GM55310).


  1. 1.
    Lohman T. M. (1993) Helicase-catalyzed DNA unwinding. J. Biol. Chem. 268, 2269–2272.PubMedGoogle Scholar
  2. 2.
    Lohman T. M., Tomko E. J., and Wu C. G. (2008) Non-hexameric DNA helicases and translocases: mechanisms and regulation. Nat. Rev. Mol. Cell Biol. 9, 391–401.PubMedCrossRefGoogle Scholar
  3. 3.
    Patel S. S. and Donmez I. (2006) Mechanisms of helicases. J. Biol. Chem. 281, 18265–18268.PubMedCrossRefGoogle Scholar
  4. 4.
    Patel S. S. and Picha K. M. (2000) Structure and function of hexameric helicases. Annu. Rev. Biochem. 69, 651–697.PubMedCrossRefGoogle Scholar
  5. 5.
    Donmez I., Rajagopal V., Jeong Y. J., and Patel S. S. (2007) Nucleic acid unwinding by hepatitis C virus and bacteriophage t7 helicases is sensitive to base pair stability. J. Biol. Chem. 282, 21116–21123.PubMedCrossRefGoogle Scholar
  6. 6.
    Stano N. M., Jeong Y. J., Donmez I., Tummalapalli P., Levin M. K., and Patel S. S. (2005) DNA synthesis provides the driving force to accelerate DNA unwinding by a helicase. Nature 435, 370–373.PubMedCrossRefGoogle Scholar
  7. 7.
    Egelman E. H., Yu X., Wild R., Hingorani M. M., and Patel S. S. (1995) Bacteriophage T7 helicase/primase proteins form rings around single-stranded DNA that suggest a general structure for hexameric helicases. Proc. Natl. Acad. Sci. USA 92, 3869–3873.PubMedCrossRefGoogle Scholar
  8. 8.
    Singleton M. R., Sawaya M. R., Ellenberger T., and Wigley D. B. (2000) Crystal structure of T7 gene 4 ring helicase indicates a mechanism for sequential hydrolysis of nucleotides. Cell 101, 589–600.PubMedCrossRefGoogle Scholar
  9. 9.
    Toth E. A., Li Y., Sawaya M. R., Cheng Y., and Ellenberger T. (2003) The crystal structure of the bifunctional primase-helicase of bacteriophage t7. Mol. Cell 12, 1113–1123.PubMedCrossRefGoogle Scholar
  10. 10.
    Tabor S. and Richardson C. C. (1981) Template recognition sequence for RNA primer synthesis by gene 4 protein of bacteriophage T7. Proc. Natl. Acad. Sci. U.S.A. 78, 205–209.PubMedCrossRefGoogle Scholar
  11. 11.
    Kim D. E., Narayan M., and Patel S. S. (2002) T7 DNA helicase: a molecular motor that processively and unidirectionally translocates along single-stranded DNA. J. Mol. Biol. 321, 807–819.PubMedCrossRefGoogle Scholar
  12. 12.
    Rasnik I., Jeong Y. J., McKinney S. A., Rajagopal V., Patel S. S., and Ha T. (2008) Branch migration enzyme as a Brownian ratchet. EMBO J. 27, 1727–1735.PubMedCrossRefGoogle Scholar
  13. 13.
    Ahnert P. and Patel S. S. (1997) Asymmetric interactions of hexameric bacteriophage T7 DNA helicase with the 5'- and 3'-tails of the forked DNA substrate. J. Biol. Chem. 272, 32267–32273.PubMedCrossRefGoogle Scholar
  14. 14.
    Hacker K. J. and Johnson K. A. (1997) A hexameric helicase encircles one DNA strand and excludes the other during DNA unwinding. Biochemistry 36, 14080–14087.PubMedCrossRefGoogle Scholar
  15. 15.
    Kaplan D. L., Davey M. J., and O’Donnell M. (2003) Mcm4,6,7 uses a ‘pump in ring’ mechanism to unwind DNA by steric exclusion and actively translocate along a duplex. J. Biol. Chem. 278, 49171–49182.PubMedCrossRefGoogle Scholar
  16. 16.
    Kaplan D. L. (2000) The 3'-tail of a forked-duplex sterically determines whether one or two DNA strands pass through the central channel of a replication-fork helicase. J. Mol. Biol. 301, 285–299.PubMedCrossRefGoogle Scholar
  17. 17.
    Jezewska M. J., Rajendran S., Bujalowska D., and Bujalowski W. (1998) Does single-stranded DNA pass through the inner channel of the protein hexamer in the complex with the Escherichia coli DnaB Helicase? Fluorescence energy transfer studies. J. Biol. Chem. 273, 10515–10529.PubMedCrossRefGoogle Scholar
  18. 18.
    Doublie S., Tabor S., Long A. M., Richardson C. C., and Ellenberger T. (1998) Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 A resolution. Nature 391, 251–258.PubMedCrossRefGoogle Scholar
  19. 19.
    Modrich P. and Richardson C. C. (1975) Bacteriophage T7 Deoxyribonucleic acid replication in vitro. A protein of Escherichia coli required for bacteriophage T7 DNA polymerase activity. J. Biol. Chem. 250, 5508–5514.PubMedGoogle Scholar
  20. 20.
    Tabor S., Huber H. E., and Richardson C. C. (1987) Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage T7. J. Biol. Chem. 262, 16212–16223.PubMedGoogle Scholar
  21. 21.
    Ha T., Rasnik I., Cheng W., Babcock H. P., Gauss G. H., Lohman T. M., and Chu S. (2002) Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase. Nature 419, 638–641.PubMedCrossRefGoogle Scholar
  22. 22.
    Dessinges M. N., Lionnet T., Xi X. G., Bensimon D., and Croquette V. (2004) Single-molecule assay reveals strand switching and enhanced processivity of UvrD. Proc. Natl. Acad. Sci. U.S.A. 101, 6439–6444.PubMedCrossRefGoogle Scholar
  23. 23.
    Dumont S., Cheng W., Serebrov V., Beran R. K., Tinoco I., Jr., Pyle A. M., and Bustamante C. (2006) RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP. Nature 439, 105–108.PubMedCrossRefGoogle Scholar
  24. 24.
    Lee J. B., Hite R. K., Hamdan S. M., Xie X. S., Richardson C. C., and van Oijen A. M. (2006) DNA primase acts as a molecular brake in DNA replication. Nature 439, 621–624.PubMedCrossRefGoogle Scholar
  25. 25.
    van Oijen A. M. (2007) Single-molecule studies of complex systems: the replisome. Mol. Biosyst. 3, 117–125.PubMedCrossRefGoogle Scholar
  26. 26.
    Johnson D. S., Bai L., Smith B. Y., Patel S. S., and Wang M. D. (2007) Single-molecule studies reveal dynamics of DNA unwinding by the ring-shaped T7 helicase. Cell 129, 1299–1309.PubMedCrossRefGoogle Scholar
  27. 27.
    van Oijen A. M. (2008) Cutting the forest to see a single tree? Nat. Chem. Biol. 4, 440–443.PubMedCrossRefGoogle Scholar
  28. 28.
    Tanner N. A., Hamdan S. M., Jergic S., Schaeffer P. M., Dixon N. E., and van Oijen A. M. (2008) Single-molecule studies of fork dynamics in Escherichia coli DNA replication. Nat. Struct. Mol. Biol. 15, 170–176.PubMedCrossRefGoogle Scholar
  29. 29.
    Lionnet T., Spiering M. M., Benkovic S. J., Bensimon D., and Croquette V. (2007) Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism. Proc. Natl. Acad. Sci. U.S.A. 104, 19790–19795.PubMedCrossRefGoogle Scholar
  30. 30.
    Ali J. A. and Lohman T. M. (1997) Kinetic measurement of the step size of DNA unwinding by Escherichia coli UvrD helicase. Science 275, 377–380.PubMedCrossRefGoogle Scholar
  31. 31.
    Jeong Y. J., Levin M. K., and Patel S. S. (2004) The DNA-unwinding mechanism of the ring helicase of bacteriophage T7. Proc. Natl. Acad. Sci. U.S.A. 101, 7264–7269.PubMedCrossRefGoogle Scholar
  32. 32.
    Picha K. M. and Patel S. S. (1998) Bacteriophage T7 DNA helicase binds dTTP, forms hexamers, and binds DNA in the absence of Mg2+. The presence of dTTP is sufficient for hexamer formation and DNA binding. J. Biol. Chem. 273, 27315–27319.PubMedCrossRefGoogle Scholar
  33. 33.
    Levin M. K., Hingorani M. H., Holmes R. M., Patel S. S. and Carson J. H. (2009) Model-based global analysis of heterogeneous experimental data using gfit. Methods Mol. Biol. 500, 335–359, Humana Press Inc.PubMedCrossRefGoogle Scholar
  34. 34.
    Patel S. S., Bandwar R. P., and Levin M. K. (2002) Transient-state kinetics and computational analysis of transcription initiation. The practical approach series/Kinetic analysis of macromolecules (Johnson K. A., Ed.), Oxford University Press, Oxford.Google Scholar
  35. 35.
    Lucius A. L., Maluf N. K., Fischer C. J., and Lohman T. M. (2003) General methods for analysis of sequential ‘n-step’ kinetic mechanisms: application to single turnover kinetics of helicase-catalyzed DNA unwinding. Biophys. J. 85, 2224–2239.PubMedCrossRefGoogle Scholar
  36. 36.
    Patel S. S., Rosenberg A. H., Studier F. W., and Johnson K. A. (1992) Large scale purification and biochemical characterization of T7 primase/helicase proteins. Evidence for homodimer and heterodimer formation. J. Biol. Chem. 267, 15013–15021.PubMedGoogle Scholar
  37. 37.
    Patel S. S., Wong I., and Johnson K. A. (1991) Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant. Biochemistry 30, 511–525.PubMedCrossRefGoogle Scholar
  38. 38.
    Lohman T. M., Green J. M., and Beyer R. S. (1986) Large-scale overproduction and rapid purification of the Escherichia coli ssb gene product. Expression of the ssb gene under lambda PL control. Biochemistry 25, 21–25.PubMedCrossRefGoogle Scholar
  39. 39.
    Donmez I. and Patel S. S. (2008) Coupling of DNA unwinding to nucleotide hydrolysis in a ring-shaped helicase. EMBO J. 27, 1718–1726.PubMedCrossRefGoogle Scholar
  40. 40.
    Cavaluzzi M. J. and Borer P. N. (2004) Revised UV extinction coefficients for nucleoside-5'-monophosphates and unpaired DNA and RNA. Nucleic Acids Res. 32, e13.PubMedCrossRefGoogle Scholar
  41. 41.
    Kallansrud G. and Ward B. (1996) A comparison of measured and calculated single- and double-stranded oligodeoxynucleotide extinction coefficients. Anal. Biochem. 236, 134–138.PubMedCrossRefGoogle Scholar
  42. 42.
    Sjoback R., Nygren J., and Kubista M. (1995) Absorption and fluorescence properties of fluorescein. Spectrochim. Acta. [A] 51, 7–21.CrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Manjula Pandey
    • 1
  • Mikhail K. Levin
    • 2
  • Smita S. Patel
    • 1
  1. 1.Depatment of BiochemistryUniversity of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical SchoolPiscatawayUSA
  2. 2.Department of Biostatistics & BioinformaticsDuke University Medical CenterDurhamUSA

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