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
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


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

PDF or HTML at Wiley Online Library

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
PDF or HTML at Wiley Online Library


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

  • 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
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Berg, J.M., Tymoczko, J.L., and Stryer, L. 2012. Enzymes: Basic concepts and kinetics. In Biochemistry, 7th ed. (L. Samols, G.L. Hadler, P. Zimmerman, and N. Brooks, eds.) pp. 229‐237. W. H. Freeman, New York.
  Biertümpfel, C., Zhao, Y., Kondo, Y., Ramón‐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 η. Nature 465:1044‐1048.
  Boosalis, M.S., Petruska, J., and Goodman, M.F. 1987. DNA polymerase insertion fidelity: Gel assay for site‐specific kinetics. J. Biol. Chem. 262:14689‐14696.
  Christov, P.P., Angel, K.C., Guengerich, F.P., and Rizzo, C.J. 2009. Replication past the N5‐methyl‐formamidopyrimidine lesion of deoxyguanosine by DNA polymerases and an improved procedure for sequence analysis of in vitro bypass products by mass spectrometry. Chem. Res. Toxicol. 22:1086‐1095.
  Dronkert, M.L. and Kanaar, R. 2001. Repair of DNA interstrand cross‐links. Mutat. Res. 486:217‐247.
  Duckett, D.R., Drummond, J.T., Murchie, A.I.H., Reardon, J.T., Sancar, A., Lilley, D.M.J., and Modrich, P. 1996. Human MutSalpha recognizes damaged DNA base pairs containing O6‐methylguanine, O4‐methylthymidine, or the cisplatin‐d(GpG) adduct. Proc. Natl. Acad. Sci. U.S.A. 93:6443‐6447.
  Ellington, A. and Pollard, J.D. 1998. Purification of oligonucleotides using denaturing polyacrylamide gel electrophoresis. Curr. Protoc. Mol. Biol. 42:2.12.1‐2.12.7.
  Guengerich, F.P. 2006. Interactions of carcinogen‐bound DNA with individual DNA polymerases. Chem. Rev. 106:420‐452.
  Hecht, S.S. 1998. Biochemistry, biology, and carcinogenicity of tobacco‐specific N‐nitrosamines. Chem. Res. Toxicol. 11:559‐603.
  Johnson, K.A. 2003. Introduction to kinetic analysis of enzyme systems. In Kinetic Analysis of Macromolecules. A Practical Approach. (K. A. Johnson, ed.) pp. 1‐18. Oxford University Press, New York.
  Kalnik, M.W., Kouchakdjian, M., Li, B.F.L., Swann, P.F., and Patel, D.J. 1988. Base pair mismatches and carcinogen‐modified bases in DNA: An NMR study of A•C and A•O4meT pairing in dodecanucleotide duplexes. Biochemistry 27:100‐108.
  Kang, H., Konishi, C., Kuroki, T., and Huh, N. 1995. Detection of O6‐methylguanine, O4‐methylthymine and O4‐ethylthymine in human liver and peripheral blood leukocyte DNA. Carcinogenesis 16:1277‐1280.
  Lawley, P.D. and Phillips, D.H. 1996. DNA adducts from chemotherapeutic. Mutat. Res. 355:13‐40.
  Lawley, P.D., Orr, D.J., Shah, S.A., Farmer, P.B., and Jarman, M. 1973. Reaction products from N‐methyl‐N‐nitrosourea and deoxyribonucleic acid containing thymidine residues. Biochem. J. 135:193‐201.
  Magaňa‐Schwencke, N., Henriques, J.‐A.P., Chanet, R., and Moustacchi, E. 1982. The fate of 8‐methoxypsoralen photoinduced crosslinks in nuclear and mitochondrial yeast DNA: Comparison of wild‐type and repair‐deficient strains. Proc. Natl. Acad. Sci. U.S.A. 79:1722‐1726.
  Michaelis, L. and Menten, M.L. 1913. Kinetik der Invertinwirkung. Biochem. Z. 49:333‐369.
  Patra, A., Nagy, L.D., Zhang, Q., Su, Y., Müller, 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 η. J. Biol. Chem. 289:16867‐16882.
  Prelich, G. and Stillman, B. 1988. Coordinated leading and lagging during SV40 DNA replication in vitro requires PCNA strand synthesis. Cell 53:117‐126.
  Samson, L., Han, S., and Marquis, J.C. 1997. Mammalian DNA repair methyltransferases shield O4MeT from nucleotide excision repair. Carcinogenesis 18:919‐924.
  Shrivastav, N., Li, D., and Essigmann, J.M. 2010. Chemical biology of mutagenesis and DNA repair: Cellular responses to DNA alkylation. Carcinogenesis 31:59‐70.
  Singer, B. 1986. O‐Alkyl pyrimidines in mutagenesis and carcinogenesis: Occurrence and significance. Cancer Res. 46:4879‐4885.
  Singer, B., Spengler, S.J., Fraenkel‐Conrat, H., and Kuśmierek, J.T. 1986. O4‐Methyl, ‐ethyl, or ‐isopropyl substituents on thymidine in poly(dA‐dT) all lead to transitions upon replication. Proc. Natl. Acad. Sci. U.S.A. 83:28‐32.
  Waga, S. and Stillman, B. 1994. Anatomy of a DNA replication fork revealed by reconstitution of SV40 DNA replication in vitro. Nature 369:207‐212.
  Zang, H., Goodenough, A.K., Choi, J.‐Y., Irimia, A., Loukachevitch, L.V, Kozekov, I.D., Angel, K.C., Rizzo, C.J., Egli, M., and Guengerich, F.P. 2005. DNA adduct bypass polymerization by Sulfolobus solfataricus DNA polymerase Dpo4: Analysis and crystal structures of multiple base pair substitution and frameshift products with the adduct 1,N2‐ethenoguanine. J. Biol. Chem. 280:29750‐29764.
PDF or HTML at Wiley Online Library