Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. 1988 Sep; 85(17): 6252–6256.
PMCID: PMC281947

Comparison between DNA melting thermodynamics and DNA polymerase fidelity.


The relation between DNA polymerase fidelity and base pairing stability is investigated by using DNA primer-template duplexes that contain a common 9-base template sequence but have either correct (A.T) or incorrect (G.T, C.T, T.T) base pairs at the primer 3' terminus. Thermal melting and enzyme kinetic measurements are compared for each kind of terminus. Analysis of melting temperatures finds that differences between the free energy changes upon dissociation (delta delta Go) are only 0.2, 0.3, and 0.4 kcal.mol-1 (1 cal = 4.18 J) for terminal A.T compared to G.T, C.T, and T.T mispairs, respectively, at 37 degrees C. We show that enthalpy changes are directly correlated with entropy changes for normal and abnormal base pairs in DNA in aqueous solution and that delta delta Go values are small because of near cancellation of corresponding enthalpy and entropy components. The kinetics of elongating primer termini are measured with purified Drosophila DNA polymerase alpha. The matched A.T terminus is found to be extended approximately 200 times faster than a G.T mismatch and 1400 and 2500 times faster than C.T and T.T mismatches, respectively. Enzymatic discrimination against elongating mismatched termini is based mainly on Km rather than Vmax differences. From Km at 37 degrees C, we find delta delta Go values of 2.6-3.7 kcal.mol-1, about an order of magnitude greater than indicated by melting data. A similar measurement of nucleotide insertion kinetics has previously found rates of forming A.T base pairs to be 500 times greater than G.T mispairs and 20,000 times greater than C.T and T.T mispairs. Here also, Km differences are mainly responsible for discrimination and indicate even larger delta delta Go values (4.3-4.9 kcal.mol-1). Thus, free energy differences between correct and incorrect base pairs in the active site cleft of polymerase appear to be greater than 10 times as large as in aqueous medium. We explore the idea that a binding cleft that snugly fits correct base pairs and excludes water at the active site may amplify base-pair free energy differences by reducing entropy differences and increasing enthalpy differences sufficiently to account for nucleotide insertion and extension fidelity.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.1M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Loeb LA, Kunkel TA. Fidelity of DNA synthesis. Annu Rev Biochem. 1982;51:429–457. [PubMed]
  • Petruska J, Sowers LC, Goodman MF. Comparison of nucleotide interactions in water, proteins, and vacuum: model for DNA polymerase fidelity. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1559–1562. [PMC free article] [PubMed]
  • Aboul-ela F, Koh D, Tinoco I, Jr, Martin FH. Base-base mismatches. Thermodynamics of double helix formation for dCA3XA3G + dCT3YT3G (X, Y = A,C,G,T). Nucleic Acids Res. 1985 Jul 11;13(13):4811–4824. [PMC free article] [PubMed]
  • Boosalis MS, Petruska J, Goodman MF. DNA polymerase insertion fidelity. Gel assay for site-specific kinetics. J Biol Chem. 1987 Oct 25;262(30):14689–14696. [PubMed]
  • Kaguni LS, Rossignol JM, Conaway RC, Lehman IR. Isolation of an intact DNA polymerase-primase from embryos of Drosophila melanogaster. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2221–2225. [PMC free article] [PubMed]
  • van Wezenbeek PM, Hulsebos TJ, Schoenmakers JG. Nucleotide sequence of the filamentous bacteriophage M13 DNA genome: comparison with phage fd. Gene. 1980 Oct;11(1-2):129–148. [PubMed]
  • Cotterill SM, Reyland ME, Loeb LA, Lehman IR. A cryptic proofreading 3'----5' exonuclease associated with the polymerase subunit of the DNA polymerase-primase from Drosophila melanogaster. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5635–5639. [PMC free article] [PubMed]
  • Breslauer KJ, Frank R, Blöcker H, Marky LA. Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3746–3750. [PMC free article] [PubMed]
  • Randall SK, Eritja R, Kaplan BE, Petruska J, Goodman MF. Nucleotide insertion kinetics opposite abasic lesions in DNA. J Biol Chem. 1987 May 15;262(14):6864–6870. [PubMed]
  • Lumry R, Rajender S. Enthalpy-entropy compensation phenomena in water solutions of proteins and small molecules: a ubiquitous property of water. Biopolymers. 1970;9(10):1125–1227. [PubMed]
  • Breslauer KJ, Remeta DP, Chou WY, Ferrante R, Curry J, Zaunczkowski D, Snyder JG, Marky LA. Enthalpy-entropy compensations in drug-DNA binding studies. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8922–8926. [PMC free article] [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...