Fidelity of DNA replication under conditions used for oligodeoxynucleotide-directed mutagenesis.
The fidelity of DNA replication in vitro by DNA polymerase I (large subfragment) of Escherichia coli has been measured by the standard bioassay: single-stranded phi X174 DNA (plus strand) containing an amber codon was primed with a synthetic oligodeoxynucleotide, replicated and the frequency of point mutations formed in the synthetic minus strand of the resultant double-stranded DNA determined from the number of revertant phage produced in a spheroplast assay. Since the assay depends crucially on the frequency of expression of the mutations in the heteroduplex, and this can vary for a variety of reasons, parallel control experiments were performed using a primer that covered the amber codon but contained the same mismatch that occurred during replication. The frequency of expression of these mutations was found to vary from 40 to 100% in fully ligated heteroduplexes, depending upon the age and batch of spheroplasts used. The variation probably reflects the viability of the post-replicative mismatch repair enzymes in the spheroplasts used for transfection. Far lower frequencies of expression were found under conditions of poor replication. Accurate data and rate laws for fidelity are obtained only when the bioassay is normalized for the variation in the expression frequency. There is active proofreading by the 3'-5'-exonuclease activity of the polymerase of a misincorporation resulting from a dGTP:T mismatch. The contribution of proofreading to fidelity is low: accuracy is enhanced by a factor of less than 7 at the concentrations of dNTPs in vivo. The lower accuracy of Pol I than Pol III is due mainly to poorer proofreading, which is manifested in a lower "cost" of replication: only 0.7 to 1.7% of the dNTPs are turned over to dNMPs during replication compared with 6 to 13% for Pol III. The error rates measured for Pol I under conditions used for oligodeoxynucleotide-directed mutagenesis are sufficiently low that extraneous errors should not be induced when the concentrations of dNTPs are balanced. However, even higher fidelity will be obtained using the lowest concentrations of dNTPs consistent with efficient replication (approximately 20 microM). Highly unbalanced concentrations as used in pulsed labelling should be avoided.
D010584:Bacteriophage phi X 174 / Q000502:physiology; D001681:Biological Assay; D004256:DNA Polymerase I / Q000378:metabolism; D004261:DNA Replication; D004270:DNA, Circular / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D014779:Virus Replication
D004270:DNA, Circular; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D004256:DNA Polymerase I