The fidelity of DNA synthesis catalyzed by derivatives of Escherichia coli DNA polymerase I.

Abstract:

The fidelity of DNA synthesis by an exonuclease-proficient DNA polymerase results from the selectivity of the polymerization reaction and from exonucleolytic proofreading. We have examined the contribution of these two steps to the fidelity of DNA synthesis catalyzed by the large Klenow fragment of Escherichia coli DNA polymerase I, using enzymes engineered by site-directed mutagenesis to inactivate the proofreading exonuclease. Measurements with two mutant Klenow polymerases lacking exonuclease activity but retaining normal polymerase activity and protein structure demonstrate that the base substitution fidelity of polymerization averages one error for each 10,000 to 40,000 bases polymerized, and can vary more than 30-fold depending on the mispair and its position. Steady-state enzyme kinetic measurements of selectivity at the initial insertion step by the exonuclease-deficient polymerase demonstrate differences in both the Km and the Vmax for incorrect versus correct nucleotides. Exonucleolytic proofreading by the wild-type enzyme improves the average base substitution fidelity by 4- to 7-fold, reflecting efficient proofreading of some mispairs and less efficient proofreading of others. The wild-type polymerase is highly accurate for -1 base frameshift errors, with an error rate of less than or equal to 10(-6). The exonuclease-deficient polymerase is less accurate, suggesting that proofreading also enhances frameshift fidelity. Even without a proofreading exonuclease, Klenow polymerase has high frameshift fidelity relative to several other DNA polymerases, including eucaryotic DNA polymerase-alpha, an exonuclease-deficient, 4-subunit complex whose catalytic subunit is almost three times larger. The Klenow polymerase has a large (46 kDa) domain containing the polymerase active site and a smaller (22 kDa) domain containing the active site for the 3'----5' exonuclease. Upon removal of the small domain, the large polymerase domain has altered base substitution error specificity when compared to the two-domain but exonuclease-deficient enzyme. It is also less accurate for -1 base errors at reiterated template nucleotides and for a 276-nucleotide deletion error. Thus, removal of a protein domain of a DNA polymerase can affect its fidelity.

Polymerases:

Topics:

Exonuclease Activity, Fidelity, Mutational Analysis, Structure and Structure/Function

One line summary:

Compares wild-type with different exo-minus mutants

Note:

Contains large amount of data on error specificity, not captured in the results summary.

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Klenow fragment Bebenek K1990 3-5' Exonuclease (proofreading) Yes
Klenow fragment Bebenek K1990 Residues Involved in Catalysis of 3-5' Exo D424, D355, E357, D501
Klenow fragment Bebenek K1990 Frameshift Error Rate 6E-07 errors/bp Technique: M13mp2 forward mutation assay (1 uM dNTP, error rate is upper limit (≤))
Klenow fragment Bebenek K1990 Nucleotide Substitution Rate 5.8E-06 errors/bp Technique: M13mp2 forward mutation assay (1 uM dNTP)
KF D424A Bebenek K1990 3-5' Exonuclease (proofreading) No
KF D424A Bebenek K1990 Frameshift Error Rate 5E-06 errors/bp Technique: M13mp2 forward mutation assay (1mM dNTP; combined with D355A,E357A results)
KF D424A Bebenek K1990 Nucleotide Substitution Rate 2.5E-05 errors/bp Technique: M13mp2 forward mutation assay (1mM dNTP; combined with D355A,E357A results)
KF D355AE357A Bebenek K1990 3-5' Exonuclease (proofreading) No
KF D355AE357A Bebenek K1990 Frameshift Error Rate 5E-06 errors/bp Technique: M13mp2 forward mutation assay (1mM dNTP; combined with D424A results)
KF D355AE357A Bebenek K1990 Nucleotide Substitution Rate 2.5E-05 errors/bp Technique: M13mp2 forward mutation assay (1mM dNTP; combined with D424A results)
KF(515-928) Bebenek K1990 3-5' Exonuclease (proofreading) No
KF(515-928) Bebenek K1990 Frameshift Error Rate 1.3E-05 errors/bp Technique: M13mp2 forward mutation assay (1mM dNTP)
KF(515-928) Bebenek K1990 Nucleotide Substitution Rate 3.2E-05 errors/bp Technique: M13mp2 forward mutation assay (1mM dNTP)
KF D424A Bebenek K1990 3-5' Exonuclease (proofreading) No
KF D424A Bebenek K1990 Frameshift Error Rate 0.0043 errors/bp Technique: Forward mutational (1 uM dNTP)
KF D424A Bebenek K1990 Nucleotide Substitution Rate 0.00012 errors/bp Technique: Reversion (1 uM dNTP)

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