Skip to main content
Plant Physiology logoLink to Plant Physiology
. 1995 Apr;107(4):1277–1284. doi: 10.1104/pp.107.4.1277

Accuracy of Deoxynucleotide Incorporation by Soybean Chloroplast DNA Polymerases Is Independent of the Presence of a 3[prime] to 5[prime] Exonuclease.

J C Bailey 2nd 1, S Heinhorst 1, G C Cannon 1
PMCID: PMC157262  PMID: 12228434

Abstract

DNA polymerase was purified from soybean (Glycine max) chloroplasts that were actively replicating DNA. The main form (form I) of the enzyme was associated with a low level of 3[prime] to 5[prime] exonuclease activity throughout purification, although the ratio of exonuclease to polymerase activity decreased with each successive purification step. A second form (form II) of DNA polymerase, which elutes from DEAE-cellulose at a higher salt concentration than form I, was devoid of any exonuclease activity. To assess the potential function of the 3[prime] to 5[prime] exonuclease in proofreading, the fidelity of deoxynucleotide incorporation was measured for form I DNA polymerase throughout purification. Despite the steadily decreasing ratio of 3[prime] to 5[prime] exonuclease to polymerase activity, the extent of misincorporation by form I enzyme remained unchanged during the final purification steps, suggesting that the exonuclease did not contribute to the accuracy of DNA synthesis by this polymerase. Fidelity of form I DNA polymerase, when compared with that of form II, revealed a higher level of misincorporation for form I enzyme, a finding that is consistent with the exonuclease playing little or no role in exonucleolytic proofreading.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

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

  1. Heinhorst S., Cannon G. C., Weissbach A. Chloroplast and mitochondrial DNA polymerases from cultured soybean cells. Plant Physiol. 1990 Apr;92(4):939–945. doi: 10.1104/pp.92.4.939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Hillebrand G. G., McCluskey A. H., Abbott K. A., Revich G. G., Beattie K. L. Misincorporation during DNA synthesis, analyzed by gel electrophoresis. Nucleic Acids Res. 1984 Apr 11;12(7):3155–3171. doi: 10.1093/nar/12.7.3155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Kaguni L. S., Olson M. W. Mismatch-specific 3'----5' exonuclease associated with the mitochondrial DNA polymerase from Drosophila embryos. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6469–6473. doi: 10.1073/pnas.86.17.6469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Keim C. A., Mosbaugh D. W. Identification and characterization of a 3' to 5' exonuclease associated with spinach chloroplast DNA polymerase. Biochemistry. 1991 Nov 19;30(46):11109–11118. doi: 10.1021/bi00110a013. [DOI] [PubMed] [Google Scholar]
  5. Kunkel T. A., Mosbaugh D. W. Exonucleolytic proofreading by a mammalian DNA polymerase. Biochemistry. 1989 Feb 7;28(3):988–995. doi: 10.1021/bi00429a011. [DOI] [PubMed] [Google Scholar]
  6. Kunkel T. A., Soni A. Exonucleolytic proofreading enhances the fidelity of DNA synthesis by chick embryo DNA polymerase-gamma. J Biol Chem. 1988 Mar 25;263(9):4450–4459. [PubMed] [Google Scholar]
  7. Longley M. J., Mosbaugh D. W. Properties of the 3' to 5' exonuclease associated with porcine liver DNA polymerase gamma. Substrate specificity, product analysis, inhibition, and kinetics of terminal excision. J Biol Chem. 1991 Dec 25;266(36):24702–24711. [PubMed] [Google Scholar]
  8. McKown R. L., Tewari K. K. Purification and properties of a pea chloroplast DNA polymerase. Proc Natl Acad Sci U S A. 1984 Apr;81(8):2354–2358. doi: 10.1073/pnas.81.8.2354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Meissner K., Heinhorst S., Cannon G. C., Börner T. Purification and characterization of a gamma-like DNA polymerase from Chenopodium album L. Nucleic Acids Res. 1993 Oct 25;21(21):4893–4899. doi: 10.1093/nar/21.21.4893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Sala F., Amileni A. R., Parisi B., Spadari S. A gamma-like DNA polymerase in spinach chloroplasts. Eur J Biochem. 1980 Nov;112(2):211–217. doi: 10.1111/j.1432-1033.1980.tb07196.x. [DOI] [PubMed] [Google Scholar]
  11. Wang T. S. Eukaryotic DNA polymerases. Annu Rev Biochem. 1991;60:513–552. doi: 10.1146/annurev.bi.60.070191.002501. [DOI] [PubMed] [Google Scholar]
  12. Wang Z. F., Yang J., Nie Z. Q., Wu M. Purification and characterization of a gamma-like DNA polymerase from Chlamydomonas reinhardtii. Biochemistry. 1991 Jan 29;30(4):1127–1131. doi: 10.1021/bi00218a034. [DOI] [PubMed] [Google Scholar]
  13. Wernette C. M., Conway M. C., Kaguni L. S. Mitochondrial DNA polymerase from Drosophila melanogaster embryos: kinetics, processivity, and fidelity of DNA polymerization. Biochemistry. 1988 Aug 9;27(16):6046–6054. doi: 10.1021/bi00416a033. [DOI] [PubMed] [Google Scholar]
  14. Wernette C. M., Kaguni L. S. A mitochondrial DNA polymerase from embryos of Drosophila melanogaster. Purification, subunit structure, and partial characterization. J Biol Chem. 1986 Nov 5;261(31):14764–14770. [PubMed] [Google Scholar]
  15. Wolfe K. H., Li W. H., Sharp P. M. Rates of nucleotide substitution vary greatly among plant mitochondrial, chloroplast, and nuclear DNAs. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9054–9058. doi: 10.1073/pnas.84.24.9054. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES