Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Mar 1;108(3):755–764. doi: 10.1083/jcb.108.3.755

The level of Z-DNA in metabolically active, permeabilized mammalian cell nuclei is regulated by torsional strain

PMCID: PMC2115406  PMID: 2921282

Abstract

Permeabilized nuclei from mammalian cells encapsulated within agarose microbeads in an isotonic buffer are active in transcription and replication (Jackson, D. A., and P. R. Cook. 1985. EMBO (Eur. Mol. Biol. Organ.) J. 4:913-918). Their DNA is intact and the nuclei are accessible to macromolecules. Myeloma nuclei prepared in this way were used to probe the extent of DNA negative supercoiling and the effects of altering torsional strain by binding radioactively labeled monoclonal antibodies to Z-DNA. Control experiments used monoclonal antibodies against a nonhistone chromosomal protein, HMG-17. On increasing the amount of anti-HMG-17 added, a binding plateau was reached encompassing a 200-fold range of antibody concentration. On binding anti-Z-DNA antibody, a similar broad plateau of constant binding was found encompassing a 100-fold range of antibody concentration. The latter result was taken as a measure of preexisting Z-DNA in the nuclei. Additional anti-Z-DNA antibody binding can be "induced" in the presence of much higher concentration of antibody, apparently by perturbing the B-DNA/Z-DNA equilibrium. On inhibiting topoisomerase I with camptothecin, an elevated antibody binding plateau was found, suggesting that elastic torsional strain in the DNA is responsible for stabilizing the preexisting Z-DNA. This interpretation is supported by the fact that addition of small, nicking amounts of DNase I leads to a complete loss of antibody binding in the Z-DNA plateau region but not in the region of "induced" Z-DNA.

Full Text

The Full Text of this article is available as a PDF (1.8 MB).

Selected References

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

  1. Azorin F., Rich A. Isolation of Z-DNA binding proteins from SV40 minichromosomes: evidence for binding to the viral control region. Cell. 1985 Jun;41(2):365–374. doi: 10.1016/s0092-8674(85)80009-x. [DOI] [PubMed] [Google Scholar]
  2. Cartwright I. L., Abmayr S. M., Fleischmann G., Lowenhaupt K., Elgin S. C., Keene M. A., Howard G. C. Chromatin structure and gene activity: the role of nonhistone chromosomal proteins. CRC Crit Rev Biochem. 1982;13(1):1–86. doi: 10.3109/10409238209108709. [DOI] [PubMed] [Google Scholar]
  3. Cook P. R. A general method for preparing intact nuclear DNA. EMBO J. 1984 Aug;3(8):1837–1842. doi: 10.1002/j.1460-2075.1984.tb02056.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dick C., Johns E. W. The effect of two acetic acid containing fixatives on the histone content of calf thymus deoxyribonucleoprotein and calf thymus tissue. Exp Cell Res. 1968 Aug-Sep;51(2-3):626–632. doi: 10.1016/0014-4827(68)90150-x. [DOI] [PubMed] [Google Scholar]
  5. Dorbic T., Wittig B. Chromatin from transcribed genes contains HMG17 only downstream from the starting point of transcription. EMBO J. 1987 Aug;6(8):2393–2399. doi: 10.1002/j.1460-2075.1987.tb02517.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dorbic T., Wittig B. Isolation of oligonucleosomes from active chromatin using HMG17-specific monoclonal antibodies. Nucleic Acids Res. 1986 Apr 25;14(8):3363–3376. doi: 10.1093/nar/14.8.3363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Drlica K., Franco R. J. Inhibitors of DNA topoisomerases. Biochemistry. 1988 Apr 5;27(7):2253–2259. doi: 10.1021/bi00407a001. [DOI] [PubMed] [Google Scholar]
  8. Hill R. J., Stollar B. D. Dependence of Z-DNA antibody binding to polytene chromosomes on acid fixation and DNA torsional strain. Nature. 1983 Sep 22;305(5932):338–340. doi: 10.1038/305338a0. [DOI] [PubMed] [Google Scholar]
  9. Jackson D. A., Cook P. R. A cell-cycle-dependent DNA polymerase activity that replicates intact DNA in chromatin. J Mol Biol. 1986 Nov 5;192(1):65–76. doi: 10.1016/0022-2836(86)90464-x. [DOI] [PubMed] [Google Scholar]
  10. Jackson D. A., Cook P. R. A general method for preparing chromatin containing intact DNA. EMBO J. 1985 Apr;4(4):913–918. doi: 10.1002/j.1460-2075.1985.tb03718.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jackson D. A., Cook P. R. Replication occurs at a nucleoskeleton. EMBO J. 1986 Jun;5(6):1403–1410. doi: 10.1002/j.1460-2075.1986.tb04374.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jackson D. A., Cook P. R. Transcription occurs at a nucleoskeleton. EMBO J. 1985 Apr;4(4):919–925. doi: 10.1002/j.1460-2075.1985.tb03719.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jaworski A., Hsieh W. T., Blaho J. A., Larson J. E., Wells R. D. Left-handed DNA in vivo. Science. 1987 Nov 6;238(4828):773–777. doi: 10.1126/science.3313728. [DOI] [PubMed] [Google Scholar]
  14. Jovin T. M., van de Sande J. H., Zarling D. A., Arndt-Jovin D. J., Eckstein F., Füldner H. H., Greider C., Grieger I., Hamori E., Kalisch B. Generation of left-handed Z-DNA in solution and visualization in polytene chromosomes by immunofluorescence. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 1):143–154. doi: 10.1101/sqb.1983.047.01.019. [DOI] [PubMed] [Google Scholar]
  15. Lafer E. M., Sousa R., Ali R., Rich A., Stollar B. D. The effect of anti-Z-DNA antibodies on the B-DNA-Z-DNA equilibrium. J Biol Chem. 1986 May 15;261(14):6438–6443. [PubMed] [Google Scholar]
  16. Lafer E. M., Sousa R., Rich A. Anti-Z-DNA antibody binding can stabilize Z-DNA in relaxed and linear plasmids under physiological conditions. EMBO J. 1985 Dec 30;4(13B):3655–3660. doi: 10.1002/j.1460-2075.1985.tb04131.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lancillotti F., Lopez M. C., Arias P., Alonso C. Z-DNA in transcriptionally active chromosomes. Proc Natl Acad Sci U S A. 1987 Mar;84(6):1560–1564. doi: 10.1073/pnas.84.6.1560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Morgenegg G., Celio M. R., Malfoy B., Leng M., Kuenzle C. C. Z-DNA immunoreactivity in rat tissues. Nature. 1983 Jun 9;303(5917):540–543. doi: 10.1038/303540a0. [DOI] [PubMed] [Google Scholar]
  19. Möller A., Gabriels J. E., Lafer E. M., Nordheim A., Rich A., Stollar B. D. Monoclonal antibodies recognize different parts of Z-DNA. J Biol Chem. 1982 Oct 25;257(20):12081–12085. [PubMed] [Google Scholar]
  20. Nordheim A., Pardue M. L., Weiner L. M., Lowenhaupt K., Scholten P., Möller A., Rich A., Stollar B. D. Analysis of Z-DNA in fixed polytene chromosomes with monoclonal antibodies that show base sequence-dependent selectivity in reactions with supercoiled plasmids and polynucleotides. J Biol Chem. 1986 Jan 5;261(1):468–476. [PubMed] [Google Scholar]
  21. Pardue M. L., Nordheim A., Lafer E. M., Stollar B. D., Rich A. Z-DNA and the polytene chromosome. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 1):171–176. doi: 10.1101/sqb.1983.047.01.022. [DOI] [PubMed] [Google Scholar]
  22. Peck L. J., Nordheim A., Rich A., Wang J. C. Flipping of cloned d(pCpG)n.d(pCpG)n DNA sequences from right- to left-handed helical structure by salt, Co(III), or negative supercoiling. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4560–4564. doi: 10.1073/pnas.79.15.4560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rich A., Nordheim A., Wang A. H. The chemistry and biology of left-handed Z-DNA. Annu Rev Biochem. 1984;53:791–846. doi: 10.1146/annurev.bi.53.070184.004043. [DOI] [PubMed] [Google Scholar]
  24. Runkel L., Nordheim A. Chemical footprinting of the interaction between left-handed Z-DNA and anti-Z-DNA antibodies by diethylpyrocarbonate carbethoxylation. J Mol Biol. 1986 Jun 5;189(3):487–501. doi: 10.1016/0022-2836(86)90319-0. [DOI] [PubMed] [Google Scholar]
  25. Singleton C. K., Klysik J., Stirdivant S. M., Wells R. D. Left-handed Z-DNA is induced by supercoiling in physiological ionic conditions. Nature. 1982 Sep 23;299(5881):312–316. doi: 10.1038/299312a0. [DOI] [PubMed] [Google Scholar]
  26. Stollar B. D. Antibodies to DNA. CRC Crit Rev Biochem. 1986;20(1):1–36. doi: 10.3109/10409238609115899. [DOI] [PubMed] [Google Scholar]
  27. Zarling D. A., Arndt-Jovin D. J., Robert-Nicoud M., McIntosh L. P., Thomae R., Jovin T. M. Immunoglobulin recognition of synthetic and natural left-handed Z DNA conformations and sequences. J Mol Biol. 1984 Jul 5;176(3):369–415. doi: 10.1016/0022-2836(84)90495-9. [DOI] [PubMed] [Google Scholar]
  28. Zhang H., Wang J. C., Liu L. F. Involvement of DNA topoisomerase I in transcription of human ribosomal RNA genes. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1060–1064. doi: 10.1073/pnas.85.4.1060. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES