Accuracy of DNA polymerase-alpha in copying natural DNA.
The fidelity of DNA polymerase-alpha from calf thymus (9S enzyme) in copying bacteriophage phi174am16 DNA in vitro has been determined from the frequency of production of different revertants. In the self-priming reaction we were able to measure the frequencies of base pairing mismatches during the course of replication on biasing the ratios of deoxynucleoside triphosphates. The frequency of dGTP:T, dGTP:G and dATP:G mismatches were 7.6 x 10(-5), 4.4 x 10(-5) and 2.8 x 10(-5), respectively, at equal concentrations of the deoxynucleoside triphosphates. dCTP:A, dGTP:A, dCTP:T and dTTP:T mismatches were below the limit of detection (<5 x 10(-6)). A synthetic dodecamer primer with a 3' end covering the first two bases of the amber codon was used to determine the misinsertion frequency of the first nucleotide incorporated. This gave a misinsertion frequency of 1.5 x 10(-4) for the dGTP:T mismatch, which is slightly higher than that observed from the pool bias studies. Further, it showed no sensitivity to biasing the nucleotide pool, suggesting a different mechanism for the incorporation of the first nucleotide. These data do not support 'energy-relay'-like models for achieving high accuracy in eukaryotes. The observed misinsertion frequencies were corrected for mismatch repair of the heteroduplexes during the transfection experiments by parallel experiments using a mismatched primer. This was synthesized to have the same G:T mismatch as produced in the preceding experiment.
Grosse
F
F
Zentrum Biochemie, Abt. Biophysikalische Chemie, Medizinische Hochschule Hannover, FRG.
Krauss
G
G
Knill-Jones
J W
JW
Fersht
A R
AR
D000818:Animals; D010584:Bacteriophage phi X 174 / Q000235:genetics / Q000378:metabolism; D020137:Base Pair Mismatch; D001483:Base Sequence; D002417:Cattle; D004256:DNA Polymerase I / Q000378:metabolism*; D017931:DNA Primers; D004261:DNA Replication / Q000502:physiology*; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D003854:Deoxyribonucleotides / Q000378:metabolism; D066298:In Vitro Techniques
D017931:DNA Primers; D004279:DNA, Viral; D003854:Deoxyribonucleotides; D004256:DNA Polymerase I