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Helicases pp 57–83Cite as

Experimental and Computational Analysis of DNA Unwinding and Polymerization Kinetics

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 587))

Abstract

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).

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Acknowledgments

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).

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Authors and Affiliations

  1. Depatment of Biochemistry, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, NJ, USA

    Manjula Pandey & Smita S. Patel

  2. Department of Biostatistics & Bioinformatics, Duke University Medical Center, Durham, NC, USA

    Mikhail K. Levin

Authors
  1. Manjula Pandey
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  2. Mikhail K. Levin
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  3. Smita S. Patel
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Editor information

Editors and Affiliations

  1. Dept. Paediatric Laboratory Medicine, Hospital for Sick Children, University Ave. 555, Toronto, M5G 1X8, Ontario, Canada

    Mohamed M. Abdelhaleem

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

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Pandey, M., Levin, M.K., Patel, S.S. (2009). Experimental and Computational Analysis of DNA Unwinding and Polymerization Kinetics. In: Abdelhaleem, M. (eds) Helicases. Methods in Molecular Biology, vol 587. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-355-8_5

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  • DOI: https://doi.org/10.1007/978-1-60327-355-8_5

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60327-354-1

  • Online ISBN: 978-1-60327-355-8

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