Fidelity of DNA polymerase I and the DNA polymerase I-DNA primase complex from Saccharomyces cerevisiae.
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.
Kunkel
T A
TA
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Hamatake
R K
RK
Motto-Fox
J
J
Fitzgerald
M P
MP
Sugino
A
A
D001483:Base Sequence; D002846:Chromatography, Affinity; D004256:DNA Polymerase I / Q000302:isolation & purification / Q000378:metabolism*; D019915:DNA Primase; D004260:DNA Repair / Q000502:physiology*; D004261:DNA Replication / Q000502:physiology*; D005092:Exonucleases / Q000032:analysis; D007164:Immunosorbents; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism; D009154:Mutation; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D013698:Templates, Genetic
D007164:Immunosorbents; D009097:Multienzyme Complexes; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004256:DNA Polymerase I; D005092:Exonucleases