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

Mol Cell Biol. 1989 Oct;9(10):4447-58. doi: 10.1128/mcb.9.10.4447.

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.

MeSH terms

  • Base Sequence
  • Chromatography, Affinity
  • DNA Polymerase I / isolation & purification
  • DNA Polymerase I / metabolism*
  • DNA Primase
  • DNA Repair / physiology*
  • DNA Replication / physiology*
  • Exonucleases / analysis
  • Immunosorbents
  • Models, Genetic
  • Molecular Sequence Data
  • Multienzyme Complexes / isolation & purification
  • Multienzyme Complexes / metabolism
  • Mutation
  • RNA Nucleotidyltransferases / metabolism*
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / physiology
  • Templates, Genetic

Substances

  • Immunosorbents
  • Multienzyme Complexes
  • DNA Polymerase I
  • DNA Primase
  • RNA Nucleotidyltransferases
  • Exonucleases