Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Dec 15;25(24):4970–4976. doi: 10.1093/nar/25.24.4970

A structure-specific endonuclease from cauliflower (Brassica oleracea var. botrytis) inflorescence.

S Kimura 1, M Kai 1, H Kobayashi 1, A Suzuki 1, H Morioka 1, E Otsuka 1, K Sakaguchi 1
PMCID: PMC147132  PMID: 9396804

Abstract

A protein with structure-specific endonuclease activity has been purified to near homogeneity from cauliflower ( Brassica oleracea var. botrytis) inflorescence through five successive column chromatographies. The protein is a single polypeptide with a molecular mass of 40 kDa. Using three different branched DNA structures (flap, pseudo-Y and stem-loop) we found that the enzyme, a cauliflower structure-specific endonuclease, cleaved the single-stranded tail in the 5'-flap and 5'-pseudo-Y structures, whereas it could not incise the 3'-flap and 3'-pseudo-Y structures. The incision points occur around the single strand-duplex junction in these DNA substrates and the enzyme leaves 5'-PO4 and 3'-OH termini on DNA. The protein also endonucleolytically cleaves on the 3'-side of the single-stranded region at the junction of unpaired and duplex DNA in the stem-loop structure. The structure-specific endonuclease activity is stimulated by Mg2+ and by Mn2+, but not by Ca2+. Like mammalian FEN-1, the protein has weak 5'-->3' double-stranded DNA-specific exonuclease activity. These results indicate that the cauliflower protein is a plant structure-specific endonuclease like mammalian FEN-1 or may be the plant alternative.

Full Text

The Full Text of this article is available as a PDF (278.9 KB).

Selected References

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

  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., Oshige M., Hirose F., Kuroda K., Matsukage A., Sakaguchi K. DNA polymerase epsilon from Drosophila melanogaster. Biochem Biophys Res Commun. 1997 Jan 13;230(2):297–301. doi: 10.1006/bbrc.1996.5945. [DOI] [PubMed] [Google Scholar]
  3. Fukata H., Fukasawa H. Isolation and partial characterization of two distinct DNA topoisomerases from cauliflower inflorescence. J Biochem. 1982 Apr;91(4):1337–1342. doi: 10.1093/oxfordjournals.jbchem.a133820. [DOI] [PubMed] [Google Scholar]
  4. Gomi K., Sakaguchi K. A new meiotic protein factor which enhances activity of meiotic DNA polymerase from Coprinus cinereus. Biochem Biophys Res Commun. 1994 Feb 15;198(3):1232–1239. doi: 10.1006/bbrc.1994.1174. [DOI] [PubMed] [Google Scholar]
  5. Habraken Y., Sung P., Prakash L., Prakash S. Structure-specific nuclease activity in yeast nucleotide excision repair protein Rad2. J Biol Chem. 1995 Dec 15;270(50):30194–30198. doi: 10.1074/jbc.270.50.30194. [DOI] [PubMed] [Google Scholar]
  6. Harrington J. J., Lieber M. R. Functional domains within FEN-1 and RAD2 define a family of structure-specific endonucleases: implications for nucleotide excision repair. Genes Dev. 1994 Jun 1;8(11):1344–1355. doi: 10.1101/gad.8.11.1344. [DOI] [PubMed] [Google Scholar]
  7. Harrington J. J., Lieber M. R. The characterization of a mammalian DNA structure-specific endonuclease. EMBO J. 1994 Mar 1;13(5):1235–1246. doi: 10.1002/j.1460-2075.1994.tb06373.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ishimi Y., Claude A., Bullock P., Hurwitz J. Complete enzymatic synthesis of DNA containing the SV40 origin of replication. J Biol Chem. 1988 Dec 25;263(36):19723–19733. [PubMed] [Google Scholar]
  9. Jacquier A., Legrain P., Dujon B. Sequence of a 10.7 kb segment of yeast chromosome XI identifies the APN1 and the BAF1 loci and reveals one tRNA gene and several new open reading frames including homologs to RAD2 and kinases. Yeast. 1992 Feb;8(2):121–132. doi: 10.1002/yea.320080207. [DOI] [PubMed] [Google Scholar]
  10. Kai M., Takahashi T., Todo T., Sakaguchi K. Novel DNA binding proteins highly specific to UV-damaged DNA sequences from embryos of Drosophila melanogaster. Nucleic Acids Res. 1995 Jul 25;23(14):2600–2607. doi: 10.1093/nar/23.14.2600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Lu B. C., Sakaguchi K. An endo-exonuclease from meiotic tissues of the basidiomycete Coprinus cinereus. Its purification and characterization. J Biol Chem. 1991 Nov 5;266(31):21060–21066. [PubMed] [Google Scholar]
  13. MacInnes M. A., Dickson J. A., Hernandez R. R., Learmonth D., Lin G. Y., Mudgett J. S., Park M. S., Schauer S., Reynolds R. J., Strniste G. F. Human ERCC5 cDNA-cosmid complementation for excision repair and bipartite amino acid domains conserved with RAD proteins of Saccharomyces cerevisiae and Schizosaccharomyces pombe. Mol Cell Biol. 1993 Oct;13(10):6393–6402. doi: 10.1128/mcb.13.10.6393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Matsuda S., Sakaguchi K., Tsukada K., Teraoka H. Characterization of DNA ligase from the fungus Coprinus cinereus. Eur J Biochem. 1996 May 1;237(3):691–697. doi: 10.1111/j.1432-1033.1996.0691p.x. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Matsunaga T., Mu D., Park C. H., Reardon J. T., Sancar A. Human DNA repair excision nuclease. Analysis of the roles of the subunits involved in dual incisions by using anti-XPG and anti-ERCC1 antibodies. J Biol Chem. 1995 Sep 1;270(35):20862–20869. doi: 10.1074/jbc.270.35.20862. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Meyerowitz E. M. Arabidopsis thaliana. Annu Rev Genet. 1987;21:93–111. doi: 10.1146/annurev.ge.21.120187.000521. [DOI] [PubMed] [Google Scholar]
  19. Murray J. M., Tavassoli M., al-Harithy R., Sheldrick K. S., Lehmann A. R., Carr A. M., Watts F. Z. Structural and functional conservation of the human homolog of the Schizosaccharomyces pombe rad2 gene, which is required for chromosome segregation and recovery from DNA damage. Mol Cell Biol. 1994 Jul;14(7):4878–4888. doi: 10.1128/mcb.14.7.4878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Sawado T., Sakaguchi K. A DNA polymerase alpha catalytic subunit is purified independently from the tissues at meiotic prometaphase I of a basidiomycete, Coprinus cinereus. Biochem Biophys Res Commun. 1997 Mar 17;232(2):454–460. doi: 10.1006/bbrc.1997.6306. [DOI] [PubMed] [Google Scholar]
  22. Scherly D., Nouspikel T., Corlet J., Ucla C., Bairoch A., Clarkson S. G. Complementation of the DNA repair defect in xeroderma pigmentosum group G cells by a human cDNA related to yeast RAD2. Nature. 1993 May 13;363(6425):182–185. doi: 10.1038/363182a0. [DOI] [PubMed] [Google Scholar]
  23. Sijbers A. M., de Laat W. L., Ariza R. R., Biggerstaff M., Wei Y. F., Moggs J. G., Carter K. C., Shell B. K., Evans E., de Jong M. C. Xeroderma pigmentosum group F caused by a defect in a structure-specific DNA repair endonuclease. Cell. 1996 Sep 6;86(5):811–822. doi: 10.1016/s0092-8674(00)80155-5. [DOI] [PubMed] [Google Scholar]
  24. Smyth D. R. Flower development. Origin of the cauliflower. Curr Biol. 1995 Apr 1;5(4):361–363. doi: 10.1016/s0960-9822(95)00072-8. [DOI] [PubMed] [Google Scholar]
  25. Sommers C. H., Miller E. J., Dujon B., Prakash S., Prakash L. Conditional lethality of null mutations in RTH1 that encodes the yeast counterpart of a mammalian 5'- to 3'-exonuclease required for lagging strand DNA synthesis in reconstituted systems. J Biol Chem. 1995 Mar 3;270(9):4193–4196. doi: 10.1074/jbc.270.9.4193. [DOI] [PubMed] [Google Scholar]
  26. Takami K., Matsuda S., Sono A., Sakaguchi K. A meiotic DNA polymerase from a mushroom, Agaricus bisporus. Biochem J. 1994 Apr 15;299(Pt 2):335–340. doi: 10.1042/bj2990335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Turchi J. J., Huang L., Murante R. S., Kim Y., Bambara R. A. Enzymatic completion of mammalian lagging-strand DNA replication. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):9803–9807. doi: 10.1073/pnas.91.21.9803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Yao M., Kow Y. W. Cleavage of insertion/deletion mismatches, flap and pseudo-Y DNA structures by deoxyinosine 3'-endonuclease from Escherichia coli. J Biol Chem. 1996 Nov 29;271(48):30672–30676. doi: 10.1074/jbc.271.48.30672. [DOI] [PubMed] [Google Scholar]

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

RESOURCES