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. 1998 Jun 1;332(Pt 2):557–563. doi: 10.1042/bj3320557

Purification and characterization of a 100 kDa DNA polymerase from cauliflower inflorescence.

H Seto 1, M Hatanaka 1, S Kimura 1, M Oshige 1, Y Tsuya 1, Y Mizushina 1, T Sawado 1, N Aoyagi 1, T Matsumoto 1, J Hashimoto 1, K Sakaguchi 1
PMCID: PMC1219513  PMID: 9601087

Abstract

A DNA polymerase from cauliflower (Brassica oleracea var. botrytis) inflorescence has been purified to near homogeneity through five successive column chromatographies, and temporally designated cauliflower polymerase 1. Cauliflower polymerase 1 is a monopolypeptide with a molecular mass of 100 kDa. The enzyme efficiently uses synthetic DNA homopolymers and moderately activated DNA and a synthetic RNA homopolymer as template-primers. The enzyme is strongly sensitive to dideoxythymidine triphosphate and N-ethylmaleimide, but it is insensitive to aphidicolin. It was stimulated with 250 mM KCl. Its mode of DNA synthesis is high-processive with or without proliferating-cell nuclear antigen. A 3'-->5' exonuclease activity is associated with cauliflower polymerase 1. The enzyme is clearly different from cauliflower mitochondrial polymerase and does not resemble the four different types of wheat DNA polymerase, designated wheat DNA polymerases A, B, CI and CII. In the present paper the role of the enzyme in plant DNA synthesis is discussed.

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Selected References

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  1. Aoyagi N., Matsuoka S., Furunobu A., Matsukage A., Sakaguchi K. Drosophila DNA polymerase delta. Purification and characterization. J Biol Chem. 1994 Feb 25;269(8):6045–6050. [PubMed] [Google Scholar]
  2. Aoyagi N., Matsuoka S., Furunobu A., Matsukage A., Sakaguchi K. Drosophila DNA polymerase delta. Purification and characterization. J Biol Chem. 1994 Feb 25;269(8):6045–6050. [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Coello P., Vázquez-Ramos J. M. Maize DNA polymerase 2 is a phosphoprotein with increasing activity during germination. Eur J Biochem. 1995 Jul 1;231(1):99–103. [PubMed] [Google Scholar]
  5. Kimura S., Kai M., Kobayashi H., Suzuki A., Morioka H., Otsuka E., Sakaguchi K. A structure-specific endonuclease from cauliflower (Brassica oleracea var. botrytis) inflorescence. Nucleic Acids Res. 1997 Dec 15;25(24):4970–4976. doi: 10.1093/nar/25.24.4970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  7. Laquel P., Litvak S., Castroviejo M. Mammalian proliferating cell nuclear antigen stimulates the processivity of two wheat embryo DNA polymerases. Plant Physiol. 1993 May;102(1):107–114. doi: 10.1104/pp.102.1.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Lee S. H., Pan Z. Q., Kwong A. D., Burgers P. M., Hurwitz J. Synthesis of DNA by DNA polymerase epsilon in vitro. J Biol Chem. 1991 Nov 25;266(33):22707–22717. [PubMed] [Google Scholar]
  9. Leem S. H., Ropp P. A., Sugino A. The yeast Saccharomyces cerevisiae DNA polymerase IV: possible involvement in double strand break DNA repair. Nucleic Acids Res. 1994 Aug 11;22(15):3011–3017. doi: 10.1093/nar/22.15.3011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Matsuda S., Takami K., Sono A., Sakaguchi K. A meiotic DNA polymerase from Coprinus cinereus: further purification and characterization. Chromosoma. 1993 Nov;102(9):631–636. doi: 10.1007/BF00352311. [DOI] [PubMed] [Google Scholar]
  11. Matsumoto T., Hata S., Suzuka I., Hashimoto J. Expression of functional proliferating-cell nuclear antigen from rice (Oryza sativa) in Escherichia coli. Activity in association with human DNA polymerase delta. Eur J Biochem. 1994 Jul 1;223(1):179–187. doi: 10.1111/j.1432-1033.1994.tb18981.x. [DOI] [PubMed] [Google Scholar]
  12. Medford J. I., Elmer J. S., Klee H. J. Molecular cloning and characterization of genes expressed in shoot apical meristems. Plant Cell. 1991 Apr;3(4):359–370. doi: 10.1105/tpc.3.4.359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Meyerowitz E. M. Arabidopsis thaliana. Annu Rev Genet. 1987;21:93–111. doi: 10.1146/annurev.ge.21.120187.000521. [DOI] [PubMed] [Google Scholar]
  15. Misumi M., Weissbach A. The isolation and characterization of DNA polymerase alpha from spinach. J Biol Chem. 1982 Mar 10;257(5):2323–2329. [PubMed] [Google Scholar]
  16. Richard M. C., Litvak S., Castroviejo M. DNA polymerase B from wheat embryos: a plant delta-like DNA polymerase. Arch Biochem Biophys. 1991 May 15;287(1):141–150. doi: 10.1016/0003-9861(91)90399-4. [DOI] [PubMed] [Google Scholar]
  17. Sakaguchi K., Boyd J. B. Purification and characterization of a DNA polymerase beta from Drosophila*. J Biol Chem. 1985 Sep 5;260(19):10406–10411. [PubMed] [Google Scholar]
  18. Sanathkumar M., Ghosh B., SenGupta D. N. Isolation of mammalian pol beta type DNA polymerase in shoot tips of germinated seedlings of IR-8 rice (Oryza sativa L.). Biochem Mol Biol Int. 1996 May;39(1):117–126. doi: 10.1080/15216549600201121. [DOI] [PubMed] [Google Scholar]
  19. Schlabach A., Fridlender B., Bolden A., Weissbach A. DNA-dependent DNA polymerases from HeLa cell nuclei. II. Template and substrate utilization. Biochem Biophys Res Commun. 1971 Aug 20;44(4):879–885. doi: 10.1016/0006-291x(71)90793-5. [DOI] [PubMed] [Google Scholar]
  20. Spanos A., Sedgwick S. G., Yarranton G. T., Hübscher U., Banks G. R. Detection of the catalytic activities of DNA polymerases and their associated exonucleases following SDS-polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Apr 24;9(8):1825–1839. doi: 10.1093/nar/9.8.1825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tsurimoto T., Melendy T., Stillman B. Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin. Nature. 1990 Aug 9;346(6284):534–539. doi: 10.1038/346534a0. [DOI] [PubMed] [Google Scholar]
  22. Weiser T., Gassmann M., Thömmes P., Ferrari E., Hafkemeyer P., Hübscher U. Biochemical and functional comparison of DNA polymerases alpha, delta, and epsilon from calf thymus. J Biol Chem. 1991 Jun 5;266(16):10420–10428. [PubMed] [Google Scholar]
  23. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]
  24. Yoo M. A., Lee W. H., Ha H. Y., Ryu J. R., Yamaguchi M., Fujikawa K., Matsukage A., Kondo S., Nishida Y. Effects of DNA polymerase beta gene over-expressed in transgenic Drosophila on DNA repair and recombination. Jpn J Genet. 1994 Feb;69(1):21–33. doi: 10.1266/jjg.69.21. [DOI] [PubMed] [Google Scholar]

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