Helicases pp 57-83 | Cite as

Experimental and Computational Analysis of DNA Unwinding and Polymerization Kinetics

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

Key words

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

Notes

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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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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