Fidelity of DNA polymerase I and the DNA polymerase I-DNA primase complex from Saccharomyces cerevisiae.

Abstract:

We have determined the fidelity of DNA synthesis by DNA polymerase I (yPol I) from Saccharomyces cerevisiae. To determine whether subunits other than the polymerase catalytic subunit influence fidelity, we measured the accuracy of yPol I purified by conventional procedures, which yields DNA polymerase with a partially proteolyzed catalytic subunit and no associated primase activity, and that of yPol I purified by immunoaffinity chromatography, which yields polymerase having a single high-molecular-weight species of the catalytic subunit, as well as three additional polypeptides and DNA primase activity. In assays that score polymerase errors within the lacZ alpha-complementation gene in M13mp2 DNA, yPol I and the yPol I-primase complex produced single-base substitutions, single-base frameshifts, and larger deletions. For specific errors and template positions, the two forms of polymerase exhibited differences in fidelity that could be as large as 10-fold. Nevertheless, results for the overall error frequency and the spectrum of errors suggest that the yPol I-DNA primase complex is not highly accurate and that, just as for the polymerase alone, its fidelity is not sufficient to account for a low spontaneous mutation rate in vivo. The specificity data also suggest models to explain -1 base frameshifts in nonrepeated sequences and certain complex deletions by a direct repeat mechanism involving aberrant loop-back synthesis.

Polymerases:

Topics:

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

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Sce Pol I Kunkel TA1989 Molecular Weight 1.8E+05 Dalton
Sce Pol I Kunkel TA1989 3-5' Exonuclease (proofreading) No
Sce Pol I Kunkel TA1989 5-3' Exonuclease Unspecified
Sce Pol I Kunkel TA1989 Frameshift Error Rate 5.88E-05 errors/bp Technique: M13mp2 forward mutation assay
Sce Pol I Kunkel TA1989 Overall Error Rate 0.000111 errors/bp Technique: M13mp2 opal codon reversion assay
Sce Pol I Kunkel TA1989 Nucleotide Substitution Rate 8.33E-05 errors/bp Technique: M13mp2 opal codon reversion assay
Sce Pol I Kunkel TA1989 Specific Activity 3.2E+05 units/mg

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