Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1984 Apr 11;12(7):3155–3171. doi: 10.1093/nar/12.7.3155

Misincorporation during DNA synthesis, analyzed by gel electrophoresis.

G G Hillebrand, A H McCluskey, K A Abbott, G G Revich, K L Beattie
PMCID: PMC318736  PMID: 6326053

Abstract

A method has been developed for simultaneous comparison of the propensity of a DNA polymerase to misincorporate at different points on a natural template-primer. In this method elongation of a [5'-32P] primer, annealed to a bacteriophage template strand, is carried out in the presence of only three dNTPs (highly purified by HPLC). Under these conditions the rate of primer elongation (monitored by gel electrophoresis/autoradiography) is limited by the rate of misincorporation at template positions complementary to the missing dNTP. Variations in the rate of elongation (revealed by autoradiographic banding patterns) reflect variations in the propensity for misincorporation at different positions along the template. The effect on primer elongation produced by addition of a chemically modified dNTP to 'minus' reactions reveals the mispairing potential of the modified nucleotide during DNA synthesis. By use of this electrophoretic assay of misincorporation we have demonstrated that the fidelity of E. coli DNA polymerase I varies greatly at different positions along a natural template, and that BrdUTP and IodUTP can be incorporated in place of dCTP during chain elongation catalyzed by this enzyme.

Full text

PDF
3155

Images in this article

3160Image
on p.3160
3162Image
on p.3162
3166Image
on p.3166

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Beattie K. L., Wiegand R. C., Radding C. M. Uptake of homologous single-stranded fragments by superhelical DNA. II. Characterization of the reaction. J Mol Biol. 1977 Nov;116(4):783–803. doi: 10.1016/0022-2836(77)90271-6. [DOI] [PubMed] [Google Scholar]
  2. Brutlag D., Kornberg A. Enzymatic synthesis of deoxyribonucleic acid. 36. A proofreading function for the 3' leads to 5' exonuclease activity in deoxyribonucleic acid polymerases. J Biol Chem. 1972 Jan 10;247(1):241–248. [PubMed] [Google Scholar]
  3. Fersht A. R. Fidelity of replication of phage phi X174 DNA by DNA polymerase III holoenzyme: spontaneous mutation by misincorporation. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4946–4950. doi: 10.1073/pnas.76.10.4946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Frank R., Köster H. DNA chain length markers and the influence of base composition on electrophoretic mobility of oligodeoxyribonucleotides in polyacrylamide-gels. Nucleic Acids Res. 1979;6(6):2069–2087. doi: 10.1093/nar/6.6.2069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hibner U., Alberts B. M. Fidelity of DNA replication catalysed in vitro on a natural DNA template by the T4 bacteriophage multi-enzyme complex. Nature. 1980 May 29;285(5763):300–305. doi: 10.1038/285300a0. [DOI] [PubMed] [Google Scholar]
  6. Kunkel T. A., Loeb L. A. On the fidelity of DNA replication. Effect of divalent metal ion activators and deoxyrionucleoside triphosphate pools on in vitro mutagenesis. J Biol Chem. 1979 Jul 10;254(13):5718–5725. [PubMed] [Google Scholar]
  7. Kunkel T. A., Loeb L. A. On the fidelity of DNA replication. The accuracy of Escherichia coli DNA polymerase I in copying natural DNA in vitro. J Biol Chem. 1980 Oct 25;255(20):9961–9966. [PubMed] [Google Scholar]
  8. Kunkel T. A., Silber J. R., Loeb L. A. The mutagenic effect of deoxynucleotide substrate imbalances during DNA synthesis with mammalian DNA polymerases. Mutat Res. 1982 Jun;94(2):413–419. doi: 10.1016/0027-5107(82)90304-9. [DOI] [PubMed] [Google Scholar]
  9. LAWLEY P. D., BROOKES P. Ionization of DNA bases or base analogues as a possible explanation of mutagenesis, with special reference to 5-bromodeoxyuridine. J Mol Biol. 1962 Mar;4:216–219. doi: 10.1016/s0022-2836(62)80053-9. [DOI] [PubMed] [Google Scholar]
  10. Moore P. D., Bose K. K., Rabkin S. D., Strauss B. S. Sites of termination of in vitro DNA synthesis on ultraviolet- and N-acetylaminofluorene-treated phi X174 templates by prokaryotic and eukaryotic DNA polymerases. Proc Natl Acad Sci U S A. 1981 Jan;78(1):110–114. doi: 10.1073/pnas.78.1.110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Reckmann B., Grosse F., Krauss G. The elongation of mismatched primers by DNA polymerase alpha from calf thymus. Nucleic Acids Res. 1983 Oct 25;11(20):7251–7260. doi: 10.1093/nar/11.20.7251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Shortle D., Grisafi P., Benkovic S. J., Botstein D. Gap misrepair mutagenesis: efficient site-directed induction of transition, transversion, and frameshift mutations in vitro. Proc Natl Acad Sci U S A. 1982 Mar;79(5):1588–1592. doi: 10.1073/pnas.79.5.1588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Strauss B., Rabkin S., Sagher D., Moore P. The role of DNA polymerase in base substitution mutagenesis on non-instructional templates. Biochimie. 1982 Aug-Sep;64(8-9):829–838. doi: 10.1016/s0300-9084(82)80138-7. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Topal M. D., Baker M. S. DNA precursor pool: a significant target for N-methyl-N-nitrosourea in C3H/10T1/2 clone 8 cells. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2211–2215. doi: 10.1073/pnas.79.7.2211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Zakour R. A., Loeb L. A. Site-specific mutagenesis by error-directed DNA synthesis. Nature. 1982 Feb 25;295(5851):708–710. doi: 10.1038/295708a0. [DOI] [PubMed] [Google Scholar]
  18. van Wezenbeek P. M., Hulsebos T. J., Schoenmakers J. G. Nucleotide sequence of the filamentous bacteriophage M13 DNA genome: comparison with phage fd. Gene. 1980 Oct;11(1-2):129–148. doi: 10.1016/0378-1119(80)90093-1. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES