Abstract
The error frequency of in vitro DNA synthesis using a natural DNA template has been measured with a biological assay for nucleotide substitutions. phiX174 DNA containing an amber mutation was copied in vitro by Escherichia coli DNA polymerase I, and the reversion frequency of the progeny DNA was determined by transfection of E. coli spheroplasts. E. coli polymerase I makes less than 1 mistake at the am3 locus for every 7700 nucleotides incorporated under standard reaction conditions. Substitution of Mn2+ for Mg2+ and unequal concentrations of deoxynucleoside triphosphate substrates raises this mutation frequency to greater than 1 in 1000. Thus, E. coli DNA polymerase I can copy natural DNA templates with high fidelity and its accuracy can be affected by alterations in reaction conditions.
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Selected References
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- Barrell B. G., Air G. M., Hutchison C. A., 3rd Overlapping genes in bacteriophage phiX174. Nature. 1976 Nov 4;264(5581):34–41. doi: 10.1038/264034a0. [DOI] [PubMed] [Google Scholar]
- Battula N., Dube D., Loeb L. A. Avian myeloblastosis virus DNA polymerase. Kinetic studies on the incorporation of noncomplementary nucleotides. J Biol Chem. 1975 Nov 10;250(21):8404–8408. [PubMed] [Google Scholar]
- Battula N., Loeb L. A. The infidelity of avian myeloblastosis virus deoxyribonucleic acid polymerase in polynucleotide replication. J Biol Chem. 1974 Jul 10;249(13):4086–4093. [PubMed] [Google Scholar]
- Chang L. M., Cassani G. R., Bollum F. J. Deoxynucleotide-polymerizing enzymes of calf thymus gland. VII. Replication of homopolymers. J Biol Chem. 1972 Dec 10;247(23):7718–7723. [PubMed] [Google Scholar]
- DEMEREC M., HANSON J. Mutagenic action of manganese chloride. Cold Spring Harb Symp Quant Biol. 1951;16:215–228. doi: 10.1101/sqb.1951.016.01.017. [DOI] [PubMed] [Google Scholar]
- Dube D. K., Loeb L. A. Manganese as a mutagenic agent during in vitro DNA synthesis. Biochem Biophys Res Commun. 1975 Dec 1;67(3):1041–1046. doi: 10.1016/0006-291x(75)90779-2. [DOI] [PubMed] [Google Scholar]
- Edgell M. H., Hutchison C. A., 3rd, Sclair M. Specific endonuclease R fragments of bacteriophage phiX174 deoxyribonucleic acid. J Virol. 1972 Apr;9(4):574–582. doi: 10.1128/jvi.9.4.574-582.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Eisenberg S., Scott J. F., Kornberg A. Enzymatic replication of viral and complementary strands of duplex DNA of phage phiX174 proceeds by seprate mechanisms. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3151–3155. doi: 10.1073/pnas.73.9.3151. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fresco J. R., Alberts B. M. THE ACCOMMODATION OF NONCOMPLEMENTARY BASES IN HELICAL POLYRIBONUCLEOTIDES AND DEOXYRIBONUCLEIC ACIDS. Proc Natl Acad Sci U S A. 1960 Mar;46(3):311–321. doi: 10.1073/pnas.46.3.311. [DOI] [PMC free article] [PubMed] [Google Scholar]
- GUTHRIE G. D., SINSHEIMER R. L. Observations on the infection of bacterial protoplasts with the deoxyribonucleic acid of bacteriophage phi X174. Biochim Biophys Acta. 1963 Jun 25;72:290–297. [PubMed] [Google Scholar]
- Gillin F. D., Nossal N. G. Control of mutation frequency by bacteriophage T4 DNA polymerase. II. Accuracy of nucleotide selection by the L88 mutator, CB120 antimutator, and wild type phage T4 DNA polymerases. J Biol Chem. 1976 Sep 10;251(17):5225–5232. [PubMed] [Google Scholar]
- Hall Z. W., Lehman I. R. An in vitro transversion by a mutationally altered T4-induced DNA polymerase. J Mol Biol. 1968 Sep 28;36(3):321–333. doi: 10.1016/0022-2836(68)90158-7. [DOI] [PubMed] [Google Scholar]
- Jovin T. M., Englund P. T., Bertsch L. L. Enzymatic synthesis of deoxyribonucleic acid. XXVI. Physical and chemical studies of a homogeneous deoxyribonucleic acid polymerase. J Biol Chem. 1969 Jun 10;244(11):2996–3008. [PubMed] [Google Scholar]
- Kallenbach N. R., Litwin S. Statistical analysis of spectral mixing curve data. J Mol Biol. 1971 Dec 28;62(3):608–611. doi: 10.1016/0022-2836(71)90159-8. [DOI] [PubMed] [Google Scholar]
- Lee A. S., Sinsheimer R. L. A cleavage map of bacteriophage phiX174 genome. Proc Natl Acad Sci U S A. 1974 Jul;71(7):2882–2886. doi: 10.1073/pnas.71.7.2882. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Loeb L. A., Springgate C. F., Battula N. Errors in DNA replication as a basis of malignant changes. Cancer Res. 1974 Sep;34(9):2311–2321. [PubMed] [Google Scholar]
- Muzyczka N., Poland R. L., Bessman M. J. Studies on the biochemical basis of spontaneous mutation. I. A comparison of the deoxyribonucleic acid polymerases of mutator, antimutator, and wild type strains of bacteriophage T4. J Biol Chem. 1972 Nov 25;247(22):7116–7122. [PubMed] [Google Scholar]
- Orgel A., Orgel L. E. Induction of mutations in bacteriophage T4 with divalent manganese. J Mol Biol. 1965 Dec;14(2):453–457. doi: 10.1016/s0022-2836(65)80195-4. [DOI] [PubMed] [Google Scholar]
- Sanger F., Air G. M., Barrell B. G., Brown N. L., Coulson A. R., Fiddes C. A., Hutchison C. A., Slocombe P. M., Smith M. Nucleotide sequence of bacteriophage phi X174 DNA. Nature. 1977 Feb 24;265(5596):687–695. doi: 10.1038/265687a0. [DOI] [PubMed] [Google Scholar]
- Schekman R. W., Iwaya M., Bromstrup K., Denhardt D. T. The mechanism of replication of phi X174 single-stranded DNA. 3. An enzymic study of the structure of the replicative form II DNA. J Mol Biol. 1971 Apr 28;57(2):177–199. doi: 10.1016/0022-2836(71)90340-8. [DOI] [PubMed] [Google Scholar]
- Sirover M. A., Loeb L. A. On the fidelity of DNA replication. Effect of metal activators during synthesis with avian myeloblastosis virus DNA polymerase. J Biol Chem. 1977 Jun 10;252(11):3605–3610. [PubMed] [Google Scholar]
- Speyer J. F. Mutagenic DNA polymerase. Biochem Biophys Res Commun. 1965 Oct 8;21(1):6–8. doi: 10.1016/0006-291x(65)90417-1. [DOI] [PubMed] [Google Scholar]
- Springgate C. F., Mildvan A. S., Abramson R., Engle J. L., Loeb L. A. Escherichia coli deoxyribonucleic acid polymerase I, a zinc metalloenzyme. Nuclear quadrupolar relaxation studies of the role of bound zinc. J Biol Chem. 1973 Sep 10;248(17):5987–5993. [PubMed] [Google Scholar]
- Sumida-Yasumoto C., Yudelevich A., Hurwitz J. DNA synthesis in vitro dependent upon phiX174 replicative form I DNA. Proc Natl Acad Sci U S A. 1976 Jun;73(6):1887–1891. doi: 10.1073/pnas.73.6.1887. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Summers J. Physical map of polyoma viral DNA fragments produced by cleavage with a restriction enzyme from Haemophilus aegyptius, endonuclease R-HaeIII. J Virol. 1975 Apr;15(4):946–953. doi: 10.1128/jvi.15.4.946-953.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- TRAUTNER T. A., SWARTZ M. N., KORNBERG A. Enzymatic synthesis of deoxyribonucleic acid. X. Influence of bromouracil substitutions on replication. Proc Natl Acad Sci U S A. 1962 Mar 15;48:449–455. doi: 10.1073/pnas.48.3.449. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Travaglini E. C., Mildvan A. S., Loeb L. A. Kinetic analysis of Escherichia coli deoxyribonucleic acid polymerase I. J Biol Chem. 1975 Nov 25;250(22):8647–8656. [PubMed] [Google Scholar]
- Weisbeek P. J., van de Pol J. H. Bological activity of phi X174 replicative form DNA fragments. Biochim Biophys Acta. 1970 Dec 14;224(2):328–338. [PubMed] [Google Scholar]