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.
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
