Steady‐State Kinetic Analysis of DNA Polymerase Single‐Nucleotide Incorporation Products

Derek K. O'Flaherty1, F. Peter Guengerich2

1 Department of Chemistry and Biochemistry, Concordia University, Montréal, Québec, 2 Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University, School of Medicine, Nashville, Tennessee
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 7.21
DOI:  10.1002/0471142700.nc0721s59
Online Posting Date:  December, 2014
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Abstract

This unit describes the experimental procedures for the steady‐state kinetic analysis of DNA synthesis across DNA nucleotides (native or modified) by DNA polymerases. In vitro primer extension experiments with a single nucleoside triphosphate species followed by denaturing polyacrylamide gel electrophoresis of the extended products is described. Data analysis procedures and fitting to steady‐state kinetic models is presented to highlight the kinetic differences involved in the bypass of damaged versus undamaged DNA. Moreover, explanations concerning problems encountered in these experiments are addressed. This approach provides useful quantitative parameters for the processing of damaged DNA by DNA polymerases. © 2014 by John Wiley & Sons, Inc.

Keywords: DNA polymerase; translesion synthesis; steady‐state kinetics

     
 
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Table of Contents

  • Basic Protocol 1: Single‐Nucleotide Incorporation Reaction of 2´‐Deoxynucleoside Triphosphates Opposite a Template Containing an O4‐Methylthymidine Insert
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Single‐Nucleotide Incorporation Reaction of 2´‐Deoxynucleoside Triphosphates Opposite a Template Containing an O4‐Methylthymidine Insert

  Materials
  • 40% acrylamide/bis‐acrylamide solution (19:1, w/w, 5% crosslinker, electrophoresis purity reagent; Bio‐Rad Laboratories)
  • Ammonium persulfate (Bio‐Rad Laboratories)
  • Aqueous ethanol (70% v/v, reagent grade)
  • Bovine serum albumin (BSA), standard solution (2 mg/mL; Pierce Protein Biology Products)
  • Bromophenol blue (Sigma‐Aldrich)
  • 2´‐Deoxyribonucleoside triphosphate solutions (dNTP, 100 mM; New England Biolabs)
  • DL‐Dithiothreitol (DTT; Research Products International)
  • DNA template (see Strategic Planning)
  • EDTA (Sigma‐Aldrich)
  • Formamide (Roche)
  • Glycerol (Sigma‐Aldrich)
  • Human DNA polymerase η (hpol η; see Strategic Planning)
  • Magnesium chloride solution (25 mM; Applied Biosystems)
  • N,N,N´,N´‐Tetramethylethylenediamine (TEMED; Bio‐Rad Laboratories)
  • Potassium chloride (KCl; Sigma‐Aldrich)
  • Primer (see Strategic Planning)
  • Quench solution (see recipe)
  • Siliconizing reagent for glass (e.g., Sigmacote; Sigma‐Aldrich)
  • Tris·Cl buffer, pH 7.5 (1 M; Trizma hydrochloride buffer solution, Sigma‐Aldrich)
  • 10× Tris‐borate‐EDTA (TBE) buffer (0.89 M Tris·Cl, 0.89 M boric acid, 20 mM EDTA; Sigma‐Aldrich)
  • Urea (electrophoresis grade, Sigma‐Aldrich)
  • Xylene cyanol FF (Sigma‐Aldrich)
  • Microcentrifuge
  • Centrifuge tube holder (UNITED Laboratory Plastics)
  • Cling plastic wrap
  • Digital timer (model # 62344‐641, VWR International)
  • 18‐G disposable needle
  • 25‐mL disposable syringe
  • Dry block heater (e.g., VWR International, model # 946310) fitted with modular heating block (e.g., VWR International, 30 wells to hold 0.65 mL centrifuge tubes) and thermometer (e.g., VWR International)
  • Flat‐end sequencing tips (0.37 mm, 1 to 200 μL; Phenix Research Products)
  • GraphPad Prism software (GraphPad Software)
  • Image J software (National Institutes of Health)
  • Lint‐free tissue (e.g., Kimwipes, Kimberly‐Clark)
  • Magnetic stir plate and stir bar
  • Microsoft Excel (Microsoft Corporation)
  • Nucleic acid electrophoresis standard vertical apparatus (e.g., Sequi‐Gen GT DNA electrophoresis cell, 38 × 50 cm; Bio‐Rad Laboratories)
  • Typhoon Trio Variable Mode Imager (GE Healthcare Life Sciences)
  • Vortex mixing device
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Figures

Videos

Literature Cited

Literature Cited
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