Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 1995 May;108(1):379–386. doi: 10.1104/pp.108.1.379

Purification and characterization of a DNA strand transferase from broccoli.

A F Tissier 1, M F Lopez 1, E R Signer 1
PMCID: PMC157344  PMID: 7784508

Abstract

A protein with DNA binding, renaturation, and strand-transfer activities has been purified to homogeneity from broccoli (Brassica oleracea var italica). The enzyme, broccoli DNA strand transferase, has a native molecular mass of at least 200 kD and an apparent subunit molecular mass of 95 kD and is isolated as a set of isoforms differing only in charge. All three activities are saturated at very low stoichiometry, one monomer per approximately 1000 nucleotides of single-stranded DNA. Strand transfer is not effected by nuclease activity and reannealing, is only slightly dependent on ATP, and is independent of added Mg2+. Transfer requires homologous single- and double-stranded DNA and at higher enzyme concentrations results in very high molecular mass complexes. As with Escherichia coli RecA, transfer by broccoli DNA strand transferase depends strongly on the presence of 3' homologous ends.

Full Text

The Full Text of this article is available as a PDF (2.3 MB).

Selected References

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

  1. Assaad F. F., Tucker K. L., Signer E. R. Epigenetic repeat-induced gene silencing (RIGS) in Arabidopsis. Plant Mol Biol. 1993 Sep;22(6):1067–1085. doi: 10.1007/BF00028978. [DOI] [PubMed] [Google Scholar]
  2. Basile G., Aker M., Mortimer R. K. Nucleotide sequence and transcriptional regulation of the yeast recombinational repair gene RAD51. Mol Cell Biol. 1992 Jul;12(7):3235–3246. doi: 10.1128/mcb.12.7.3235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Binet M. N., Osman M., Jagendorf A. T. Genomic nucleotide sequence of a gene from Arabidopsis thaliana encoding a protein homolog of Escherichia coli RecA. Plant Physiol. 1993 Oct;103(2):673–674. doi: 10.1104/pp.103.2.673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bishop D. K., Park D., Xu L., Kleckner N. DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression. Cell. 1992 May 1;69(3):439–456. doi: 10.1016/0092-8674(92)90446-j. [DOI] [PubMed] [Google Scholar]
  5. Cerutti H., Jagendorf A. T. DNA Strand-Transfer Activity in Pea (Pisum sativum L.) Chloroplasts. Plant Physiol. 1993 May;102(1):145–153. doi: 10.1104/pp.102.1.145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cerutti H., Osman M., Grandoni P., Jagendorf A. T. A homolog of Escherichia coli RecA protein in plastids of higher plants. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8068–8072. doi: 10.1073/pnas.89.17.8068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen J., Kanaar R., Cozzarelli N. R. The Sep1 strand exchange protein from Saccharomyces cerevisiae promotes a paranemic joint between homologous DNA molecules. Genes Dev. 1994 Jun 1;8(11):1356–1366. doi: 10.1101/gad.8.11.1356. [DOI] [PubMed] [Google Scholar]
  8. Clark A. B., Dykstra C. C., Sugino A. Isolation, DNA sequence, and regulation of a Saccharomyces cerevisiae gene that encodes DNA strand transfer protein alpha. Mol Cell Biol. 1991 May;11(5):2576–2582. doi: 10.1128/mcb.11.5.2576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dykstra C. C., Hamatake R. K., Sugino A. DNA strand transfer protein beta from yeast mitotic cells differs from strand transfer protein alpha from meiotic cells. J Biol Chem. 1990 Jul 5;265(19):10968–10973. [PubMed] [Google Scholar]
  10. FRASER D., JERREL E. A. The amino acid composition of T3 bacteriophage. J Biol Chem. 1953 Nov;205(1):291–295. [PubMed] [Google Scholar]
  11. Halbrook J., McEntee K. Purification and characterization of a DNA-pairing and strand transfer activity from mitotic Saccharomyces cerevisiae. J Biol Chem. 1989 Dec 15;264(35):21403–21412. [PubMed] [Google Scholar]
  12. Hotta Y., Tabata S., Bouchard R. A., Piñon R., Stern H. General recombination mechanisms in extracts of meiotic cells. Chromosoma. 1985;93(2):140–151. doi: 10.1007/BF00293161. [DOI] [PubMed] [Google Scholar]
  13. Johnson A. W., Kolodner R. D. Strand exchange protein 1 from Saccharomyces cerevisiae. A novel multifunctional protein that contains DNA strand exchange and exonuclease activities. J Biol Chem. 1991 Jul 25;266(21):14046–14054. [PubMed] [Google Scholar]
  14. Judd S. R., Petes T. D. Physical lengths of meiotic and mitotic gene conversion tracts in Saccharomyces cerevisiae. Genetics. 1988 Mar;118(3):401–410. doi: 10.1093/genetics/118.3.401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kieber J. J., Lopez M. F., Tissier A. F., Signer E. Purification and properties of DNA topoisomerase I from broccoli. Plant Mol Biol. 1992 Mar;18(5):865–871. doi: 10.1007/BF00019201. [DOI] [PubMed] [Google Scholar]
  16. Kieber J. J., Tissier A. F., Signer E. R. Cloning and Characterization of an Arabidopsis thaliana Topoisomerase I Gene. Plant Physiol. 1992 Aug;99(4):1493–1501. doi: 10.1104/pp.99.4.1493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kim J., Ljungdahl P. O., Fink G. R. kem mutations affect nuclear fusion in Saccharomyces cerevisiae. Genetics. 1990 Dec;126(4):799–812. doi: 10.1093/genetics/126.4.799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kipling D., Kearsey S. E. TFIIS and strand-transfer proteins. Nature. 1991 Oct 10;353(6344):509–509. doi: 10.1038/353509a0. [DOI] [PubMed] [Google Scholar]
  19. Kipling D., Tambini C., Kearsey S. E. rar mutations which increase artificial chromosome stability in Saccharomyces cerevisiae identify transcription and recombination proteins. Nucleic Acids Res. 1991 Apr 11;19(7):1385–1391. doi: 10.1093/nar/19.7.1385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kmiec E., Holloman W. K. Homologous pairing of DNA molecules promoted by a protein from Ustilago. Cell. 1982 Jun;29(2):367–374. doi: 10.1016/0092-8674(82)90153-2. [DOI] [PubMed] [Google Scholar]
  21. Kodadek T., Wong M. L., Alberts B. M. The mechanism of homologous DNA strand exchange catalyzed by the bacteriophage T4 uvsX and gene 32 proteins. J Biol Chem. 1988 Jul 5;263(19):9427–9436. [PubMed] [Google Scholar]
  22. Kolodner R., Evans D. H., Morrison P. T. Purification and characterization of an activity from Saccharomyces cerevisiae that catalyzes homologous pairing and strand exchange. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5560–5564. doi: 10.1073/pnas.84.16.5560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Konforti B. B., Davis R. W. ATP hydrolysis and the displaced strand are two factors that determine the polarity of RecA-promoted DNA strand exchange. J Mol Biol. 1992 Sep 5;227(1):38–53. doi: 10.1016/0022-2836(92)90680-i. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Liu Z., Gilbert W. The yeast KEM1 gene encodes a nuclease specific for G4 tetraplex DNA: implication of in vivo functions for this novel DNA structure. Cell. 1994 Jul 1;77(7):1083–1092. doi: 10.1016/0092-8674(94)90447-2. [DOI] [PubMed] [Google Scholar]
  26. Lowenhaupt K., Sander M., Hauser C., Rich A. Drosophila melanogaster strand transferase. A protein that forms heteroduplex DNA in the absence of both ATP and single-strand DNA binding protein. J Biol Chem. 1989 Dec 5;264(34):20568–20575. [PubMed] [Google Scholar]
  27. McEntee K., Weinstock G. M., Lehman I. R. recA protein-catalyzed strand assimilation: stimulation by Escherichia coli single-stranded DNA-binding protein. Proc Natl Acad Sci U S A. 1980 Feb;77(2):857–861. doi: 10.1073/pnas.77.2.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Moore S. P., Fishel R. Purification and characterization of a protein from human cells which promotes homologous pairing of DNA. J Biol Chem. 1990 Jul 5;265(19):11108–11117. [PubMed] [Google Scholar]
  29. Patton W. F., Pluskal M. G., Skea W. M., Buecker J. L., Lopez M. F., Zimmermann R., Belanger L. M., Hatch P. D. Development of a dedicated two-dimensional gel electrophoresis system that provides optimal pattern reproducibility and polypeptide resolution. Biotechniques. 1990 May;8(5):518–527. [PubMed] [Google Scholar]
  30. Rosselli W., Stasiak A. The ATPase activity of RecA is needed to push the DNA strand exchange through heterologous regions. EMBO J. 1991 Dec;10(13):4391–4396. doi: 10.1002/j.1460-2075.1991.tb05017.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sander M., Lowenhaupt K., Rich A. Drosophila Rrp1 protein: an apurinic endonuclease with homologous recombination activities. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6780–6784. doi: 10.1073/pnas.88.15.6780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Shinohara A., Ogawa H., Ogawa T. Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein. Cell. 1992 May 1;69(3):457–470. doi: 10.1016/0092-8674(92)90447-k. [DOI] [PubMed] [Google Scholar]
  33. Sugino A., Nitiss J., Resnick M. A. ATP-independent DNA strand transfer catalyzed by protein(s) from meiotic cells of the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3683–3687. doi: 10.1073/pnas.85.11.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tishkoff D. X., Johnson A. W., Kolodner R. D. Molecular and genetic analysis of the gene encoding the Saccharomyces cerevisiae strand exchange protein Sep1. Mol Cell Biol. 1991 May;11(5):2593–2608. doi: 10.1128/mcb.11.5.2593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tsang S. S., Chow S. A., Radding C. M. Networks of DNA and RecA protein are intermediates in homologous pairing. Biochemistry. 1985 Jun 18;24(13):3226–3232. doi: 10.1021/bi00334a023. [DOI] [PubMed] [Google Scholar]
  36. Weinstock G. M., McEntee K., Lehman I. R. ATP-dependent renaturation of DNA catalyzed by the recA protein of Escherichia coli. Proc Natl Acad Sci U S A. 1979 Jan;76(1):126–130. doi: 10.1073/pnas.76.1.126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. West S. C. Enzymes and molecular mechanisms of genetic recombination. Annu Rev Biochem. 1992;61:603–640. doi: 10.1146/annurev.bi.61.070192.003131. [DOI] [PubMed] [Google Scholar]
  38. Yonesaki T., Minagawa T. T4 phage gene uvsX product catalyzes homologous DNA pairing. EMBO J. 1985 Dec 1;4(12):3321–3327. doi: 10.1002/j.1460-2075.1985.tb04083.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES