Experimental and computational analysis of DNA unwinding and polymerization kinetics

Manjula Pandey, Mikhail K. Levin, Smita S. Patel

Research output: Chapter in Book/Report/Conference proceedingChapter

8 Scopus citations

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 ).

Original languageEnglish (US)
Title of host publicationHelicases
Subtitle of host publicationMethods and Protocols
Pages57-83
Number of pages27
DOIs
StatePublished - Dec 1 2010

Publication series

NameMethods in Molecular Biology
Volume587
ISSN (Print)1064-3745

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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  • Cite this

    Pandey, M., Levin, M. K., & Patel, S. S. (2010). Experimental and computational analysis of DNA unwinding and polymerization kinetics. In Helicases: Methods and Protocols (pp. 57-83). (Methods in Molecular Biology; Vol. 587). https://doi.org/10.1007/978-1-60327-355-8_5