Fidelity of DNA synthesis catalyzed by human DNA polymerase alpha and HIV-1 reverse transcriptase: effect of reaction pH.

Abstract:

The accuracy of DNA synthesis catalyzed by the Thermus aquaticus DNA polymerase and the 3'-->5' exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I varies as a function of reaction pH (Eckert, K.A. and Kunkel, T.A. (1990) Nucleic Acids Res. 18, 3739-3744; Eckert, K.A. and Kunkel, T.A. (1993) J. Biol. Chem. 268, 13462-13471). In the current study, we demonstrate that the fidelity of human DNA polymerase alpha increases 10-fold when the pH of the in vitro synthesis reaction is lowered from pH 8.6 to pH 6.1 (37 degrees C), as determined using a base substitution reversion assay to score polymerase errors within the lacZ alpha gene of bacteriophage M13mp2. Similarly, the base substitution fidelity of DNA-dependent DNA synthesis by the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) was improved nine-fold at pH 6.5 relative to pH 8.0 (37 degrees C). A detailed comparison of HIV-1 RT error specificity at neutral and low pH in a lacZ alpha forward mutation assay revealed that low pH suppresses both mispairing-mediated and misalignment-mediated mutations; however, the characteristic HIV-1 RT pattern of mutational hotspots at homopolymeric sequences is retained at the lower pH. Consistent with the presumption that these mutations result, in part, from increased termination of DNA synthesis within the hotspot sequences relative to other homopolymeric sequences, the HIV-1 RT termination pattern during processive DNA synthesis is not altered by low pH. The HIV-1 RT results are in agreement with our previous hypothesis that the observed increase in polymerase fidelity at low pH results from a decreased efficiency of continuing DNA synthesis from premutational DNA intermediates.

Polymerases:

Topics:

Historical Protein Properties (MW, pI, ...), Fidelity, Exonuclease Activity, Source / Purification

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
HIV RT Eckert KA1993 3-5' Exonuclease (proofreading) No
HIV RT Eckert KA1993 Cloned or native Cloned in E. coli
HIV RT Eckert KA1993 Nucleotide Substitution Rate 2.63E-05 errors/bp Technique: Reversion (pH 6.5)
HIV RT Eckert KA1993 Nucleotide Substitution Rate 0.00027 errors/bp Technique: Reversion (pH 8.0)
HIV RT Eckert KA1993 Full length or truncated Full length
HIV RT Eckert KA1993 Specific Activity 1 umol/min/mg
Human Pol alpha Eckert KA1993 Cloned or native Insect cells
Human Pol alpha Eckert KA1993 Nucleotide Substitution Rate 2.38E-05 errors/bp Technique: Reversion (pH 6.1)
Human Pol alpha Eckert KA1993 Nucleotide Substitution Rate 0.000154 errors/bp Technique: Reversion (pH 7.8)
Human Pol alpha Eckert KA1993 Nucleotide Substitution Rate 0.000256 errors/bp Technique: Reversion (pH 8.6)
Human Pol alpha Eckert KA1993 Full length or truncated Full length
Human Pol alpha Eckert KA1993 Specific Activity 3.27E+04 units/mg

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