Advertisement

Determining Steady-State Kinetics of DNA Polymerase Nucleotide Incorporation

  • Hailey L. Gahlon
  • Shana J. SturlaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1973)

Abstract

Polymerase enzymes catalyze the replication of DNA by incorporating deoxynucleoside monophosphates (dNMPs) into a primer strand in a 5′ to 3′ direction. Monitoring kinetic aspects of this catalytic process provides mechanistic information regarding polymerase-mediated DNA synthesis and the influences of nucleobase structure. For example, a range of polymerases have different capacities to synthesize DNA depending on the structure of the inserted dNMP (natural or synthetic) and also depending on the templating DNA base (modified vs. unmodified). Under steady-state conditions, relative rates depend on the deoxynucleoside triphosphate (dNTP) residence times in the ternary (polymerase-DNA-dNTP) complex. This chapter describes a method to measure steady-state incorporation efficiencies by which polymerase enzymes insert dNMPs into primer-template (P/T) oligonucleotides. The method described involves the use of a primer oligonucleotide 5′ radiolabeled with [γ-32P]ATP. Significant established applications of this experiment include studies regarding mechanisms of nucleotide misincorporation as a basis of chemically induced DNA mutation. Further, it can provide information important in various contexts ranging from biophysical to medical-based studies.

Key words

DNA polymerase Steady-state enzyme kinetics Primer extension assay Nucleoside triphosphates 

Notes

Acknowledgments

This work was supported by the European Research Council (260341) and the Swiss National Science Foundation (156280). We would like to thank Professors F. Peter Guengerich and Robert Eoff for sharing with us their knowledge and technical expertise of the experiments described here.

References

  1. 1.
    Bloom LB, Chen X, Fygenson DK, Turner J, O’Donnell M, Goodman MF (1997) Fidelity of Escherichia coli DNA polymerase III holoenzyme. The effects of beta, gamma complex processivity proteins and epsilon proofreading exonuclease on nucleotide misincorporation efficiencies. J Biol Chem 272(44):27919–27930CrossRefGoogle Scholar
  2. 2.
    Kunkel TA, Bebenek K (2000) DNA replication fidelity. Annu Rev Biochem 69(1):497–529CrossRefGoogle Scholar
  3. 3.
    Hirao I, Mitsui T, Kimoto M, Yokoyama S (2007) An efficient unnatural base pair for PCR amplification. J Am Chem Soc 129(50):15549–15555CrossRefGoogle Scholar
  4. 4.
    Eoff RL, Stafford JB, Szekely J, Rizzo CJ, Egli M, Guengerich FP, Marnett LJ (2009) Structural and functional analysis of sulfolobus solfataricus Y-family DNA polymerase Dpo4-catalyzed bypass of the malondialdehyde−deoxyguanosine adduct. Biochemistry 48(30):7079–7088CrossRefGoogle Scholar
  5. 5.
    Gahlon HL, Schweizer WB, Sturla SJ (2013) Tolerance of base pair size and shape in postlesion DNA synthesis. J Am Chem Soc 135(17):6384–6387CrossRefGoogle Scholar
  6. 6.
    Gahlon HL, Boby ML, Sturla SJ (2014) O6-alkylguanine postlesion DNA synthesis is correct with the right complement of hydrogen bonding. ACS Chem Biol 9(12):2807–2814CrossRefGoogle Scholar
  7. 7.
    Dahlmann HA, Vaidyanathan VG, Sturla SJ (2009) Investigating the biochemical impact of DNA damage with structure-based probes: abasic sites, photodimers, alkylation adducts, and oxidative lesions. Biochemistry 48(40):9347–9359CrossRefGoogle Scholar
  8. 8.
    Wyss LA, Nilforoushan A, Eichenseher F, Suter U, Blatter N, Marx A, Sturla SJ (2015) Specific incorporation of an artificial nucleotide opposite a mutagenic DNA adduct by a DNA polymerase. J Am Chem Soc 137(1):30–33CrossRefGoogle Scholar
  9. 9.
    Berdis AJ (2009) Mechanisms of DNA polymerases. Chem Rev 109(7):2862–2879CrossRefGoogle Scholar
  10. 10.
    Boosalis MS, Petruska J, Goodman MF (1987) DNA polymerase insertion fidelity. Gel assay for site-specific kinetics. J Biol Chem 262(30):14689–14696PubMedGoogle Scholar
  11. 11.
    O’Flaherty DK, Guengerich FP (2014) Steady-state kinetic analysis of DNA polymerase single-nucleotide incorporation products. Curr Protoc Nucleic Acid Chem 59:7.21.1–7.21.13CrossRefGoogle Scholar
  12. 12.
    Schermerhorn KM, Gardner AF (2015) Pre-steady-state kinetic analysis of a family D DNA polymerase from Thermococcus sp. 9 degrees N reveals mechanisms for archaeal genomic replication and maintenance. J Biol Chem 290(36):21800–21810CrossRefGoogle Scholar
  13. 13.
    Lahiri I, Mukherjee P, Pata JD (2013) Kinetic characterization of exonuclease-deficient Staphylococcus aureus PolC, a C-family replicative DNA polymerase. PLoS One 8(5):e63489CrossRefGoogle Scholar
  14. 14.
    Guengerich FP (2006) Interactions of carcinogen-bound DNA with individual DNA polymerases. Chem Rev 106(2):420–452CrossRefGoogle Scholar
  15. 15.
    Sambrook J, Russell DW (2001) Molecular cloning a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Health Sciences and TechnologyETH ZurichZurichSwitzerland

Personalised recommendations