Pre‐Steady‐State Kinetic Analysis of Single‐Nucleotide Incorporation by DNA Polymerases

Yan Su1, F. Peter Guengerich1

1 Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 7.23
DOI:  10.1002/cpnc.2
Online Posting Date:  June, 2016
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Pre‐steady‐state kinetic analysis is a powerful and widely used method to obtain multiple kinetic parameters. This protocol provides a step‐by‐step procedure for pre‐steady‐state kinetic analysis of single‐nucleotide incorporation by a DNA polymerase. It describes the experimental details of DNA substrate annealing, reaction mixture preparation, handling of the RQF‐3 rapid quench‐flow instrument, denaturing polyacrylamide DNA gel preparation, electrophoresis, quantitation, and data analysis. The core and unique part of this protocol is the rationale for preparation of the reaction mixture (the ratio of the polymerase to the DNA substrate) and methods for conducting pre‐steady‐state assays on an RQF‐3 rapid quench‐flow instrument, as well as data interpretation after analysis. In addition, the methods for the DNA substrate annealing and DNA polyacrylamide gel preparation, electrophoresis, quantitation and analysis are suitable for use in other studies. © 2016 by John Wiley & Sons, Inc.

Keywords: DNA polymerase; pre‐steady‐state kinetics

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • DNA primer: 5′‐/FAM/CGG GCT CGT AAG CGT CAT‐3′ (Integrated DNA Technologies)
  • Nuclease‐free H 2O
  • DNA template: 5′‐TCA T(8‐oxodG)A TGA CGC TTA CGA GCC CG‐3′ (Integrated DNA Technologies)
  • Tris·Cl, pH 7.5
  • EDTA
  • Methanol
  • Ice
  • Gel loading buffer (see recipe)
  • 40% Acrylamide/bis, 19:1, w/v, 5% cross‐linker (Bio‐Rad Laboratories)
  • 5× TBE buffer (0.445 M Tris·Cl, 0.445 M boric acid, and 0.01 M EDTA)
  • Urea (electrophoresis grade, Sigma‐Aldrich)
  • Ammonium persulfate (APS; Bio‐Rad Laboratories)
  • N,N,N′,N′‐Tetramethylethylenediamine (TEMED; Bio‐Rad Laboratories)
  • Sequi‐Gen GT Nucleic Acid Sequencing Cell, 38 × 50 cm (Bio‐Rad Laboratories)
  • Dry block heater and thermometer (VWR International)
  • 1.5‐mL microcentrifuge tubes
  • Microcentrifuge
  • Water bath
  • RQF‐3 Rapid Quench‐Flow Instrument (KinTek Corporation)
  • Luer Lock disposable syringes, 1 mL and 5 mL (Bio‐Rad Laboratories)
  • Plastic cling wrap
  • Typhoon System (GE Healthcare Life Sciences)
  • ImageJ software (National Institutes of Health)
  • GraphPad Prism software (GraphPad)
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Literature Cited

Literature Cited
  Beckman, J.W., Wang, Q., and Guengerich, F.P. 2008. Kinetic analysis of correct nucleotide insertion by a Y‐family DNA polymerase reveals conformational changes both prior to and following phosphodiester bond formation as detected by tryptophan fluorescence. J. Biol. Chem. 283:36711‐36723 doi: 10.1074/jbc.M806785200.
  Biertumpfel, C., Zhao, Y., Kondo, Y., Ramon‐Maiques, S., Gregory, M., Lee, J.Y., Masutani, C., Lehmann, A.R., Hanaoka, F., and Yang, W. 2010. Structure and mechanism of human DNA polymerase eta. Nature 465:1044‐1048 doi: 10.1038/nature09196.
  Furge, L.L. and Guengerich, F.P. 1999. Explanation of pre‐steady‐state kinetics and decreased burst amplitude of HIV‐1 reverse transcriptase at sites of modified DNA bases with an additional, nonproductive enzyme‐DNA‐nucleotide complex. Biochemistry 38:4818‐4825 doi: 10.1021/bi982163u.
  Guengerich, F.P. 2006. Interactions of carcinogen‐bound DNA with individual DNA polymerases. Chem. Rev. 106:420‐452 doi: 10.1021/cr0404693.
  Johnson, K.A. 1986. Rapid kinetic analysis of mechanochemical adenosinetriphosphatases. Methods Enzymol. 134:677‐705. doi: 10.1016/0076‐6879(86)34129‐6.
  Johnson, K.A. 1993. Conformational coupling in DNA polymerase fidelity. Annu. Rev. Biochem. 62:685‐713 doi: 10.1146/
  Johnson, K.A. 1995. Rapid quench kinetic analysis of polymerases, adenosinetriphosphatases, and enzyme intermediates. Methods Enzymol. 249:38‐61. doi: 10.1016/0076‐6879 (95)49030‐2.
  Johnson, K.A. 1998. Advances in transient‐state kinetics. Curr. Opin. Biotechnol. 9:87‐89. doi: 10.1016/S0958‐1669(98)80089‐X.
  O'Flaherty, D. K. and Guengerich, F. P. 2014. Steady‐State Kinetic Analysis of DNA Polymerase Single‐Nucleotide Incorporation Products. Curr. Protoc. Nucleic Acid Chem. 59:7.21.1‐7.21.13. doi: 10.1002/0471142700.nc0721s59
  Patra, A., Zhang, Q., Su, Y., Egli, M., and Guengerich, F.P. 2015. Structural and kinetic analysis of nucleoside triphosphate incorporation opposite an abasic site by human translesion DNA polymerase h. J. Biol. Chem. 290:8028‐8038. doi: 10.1074/jbc.M115.637561.
  Patra, A., Nagy, L.D., Zhang, Q., Su, Y., Muller, L., Guengerich, F.P., and Egli, M. 2014. Kinetics, structure, and mechanism of 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine bypass by human DNA polymerase h. J. Biol. Chem. 289:16867‐16882. doi: 10.1074/jbc.M114.551820.
  Sassa, A., Beard, W.A., Shock, D.D., and Wilson, S.H. 2013. Steady‐state, pre‐steady‐state, and single‐turnover kinetic measurement for DNA glycosylase activity. J. Visualized Expts. e50695.
  Su, Y., Patra, A., Harp, J.M., Egli, M., and Guengerich, F.P. 2015. Roles of residues Arg‐61 and Gln‐38 of human DNA polymerase η in bypass of deoxyguanosine and 7,8‐dihydro‐8‐oxo‐2′‐deoxyguanosine. J. Biol. Chem. 290:15921‐15933. doi: 10.1074/jbc.M115.653691.
  Zhao, L., Pence, M.G., Eoff, R.L., Yuan, S., Fercu, C.A., and Guengerich, F.P. 2014. Elucidation of kinetic mechanisms of human translesion DNA polymerase k using tryptophan mutants. FEBS J. 281:4394‐4410. doi: 10.1111/febs.12947.
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