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. 1985 Mar;4(3):837–844. doi: 10.1002/j.1460-2075.1985.tb03706.x

Electron microscopy of SV40 DNA cross-linked by anti-Z DNA IgG.

F K Hagen, D A Zarling, T M Jovin
PMCID: PMC554265  PMID: 2988937

Abstract

Electron microscopy has revealed the specific binding of bivalent anti-Z DNA immunoglobulin G (IgG) to different sites on supercoiled Form I SV40 DNA. The anti-Z IgG links together left-handed regions located within individual or on multiple SV40 DNA molecules at the superhelix density obtained upon extraction. Velocity sedimentation, electrophoresis, and electron microscopy all show that two or more Z DNA sites in the SV40 genome can be intermolecularly cross-linked with bivalent IgG into high mol. wt. complexes. The formation and stability of the intermolecular antibody-DNA complexes are dependent on DNA superhelix density, as judged by three criteria: (1) relaxed circular (Form II) DNA does not react; (2) release of torsional stress by intercalation of 0.25 microM ethidium bromide removes the antibody; and (3) linearization with specific restriction endonucleases reverses antibody binding and DNA cross-linking. Non-immune IgG does not bind to negatively supercoiled SV40 Form I DNA, nor are complexes observed in the presence of competitive synthetic polynucleotides constitutively in the left-handed Z conformation; B DNA has no effect. Using various restriction endonucleases, three major sites of anti-Z IgG binding have been mapped by electron microscopy to the 300-bp region containing nucleotide sequences controlling SV40 gene expression. A limited number of minor sites may also exist (at the extracted superhelix density).

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Selected References

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

  1. Di Capua E., Stasiak A., Koller T., Brahms S., Thomae R., Pohl F. M. Torsional stress induces left-handed helical stretches in DNA of natural base sequence: circular dichroism and antibody binding. EMBO J. 1983;2(9):1531–1535. doi: 10.1002/j.1460-2075.1983.tb01619.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  3. Jovin T. M., McIntosh L. P., Arndt-Jovin D. J., Zarling D. A., Robert-Nicoud M., van de Sande J. H., Jorgenson K. F., Eckstein F. Left-handed DNA: from synthetic polymers to chromosomes. J Biomol Struct Dyn. 1983 Oct;1(1):21–57. doi: 10.1080/07391102.1983.10507425. [DOI] [PubMed] [Google Scholar]
  4. Konopka A. K., Reiter J., Jung M., Zarling D. A., Jovin T. M. Concordance of experimentally mapped or predicted Z-DNA sites with positions of selected alternating purine-pyrimidine tracts. Nucleic Acids Res. 1985 Mar 11;13(5):1683–1701. doi: 10.1093/nar/13.5.1683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Nordheim A., Rich A. Negatively supercoiled simian virus 40 DNA contains Z-DNA segments within transcriptional enhancer sequences. Nature. 1983 Jun 23;303(5919):674–679. doi: 10.1038/303674a0. [DOI] [PubMed] [Google Scholar]
  6. Pohl F. M., Jovin T. M., Baehr W., Holbrook J. J. Ethidium bromide as a cooperative effector of a DNA structure. Proc Natl Acad Sci U S A. 1972 Dec;69(12):3805–3809. doi: 10.1073/pnas.69.12.3805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Revet B., Zarling D. A., Jovin T. M., Delain E. Different Z DNA forming sequences are revealed in phi X174 RFI by high resolution darkfield immuno-electron microscopy. EMBO J. 1984 Dec 20;3(13):3353–3358. doi: 10.1002/j.1460-2075.1984.tb02303.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Rich A., Nordheim A., Azorin F. Stabilization and detection of natural left-handed Z-DNA. J Biomol Struct Dyn. 1983 Oct;1(1):1–19. doi: 10.1080/07391102.1983.10507424. [DOI] [PubMed] [Google Scholar]
  9. Robert-Nicoud M., Arndt-Jovin D. J., Zarling D. A., Jovin T. M. Immunological detection of left-handed Z DNA in isolated polytene chromosomes. Effects of ionic strength, pH, temperature and topological stress. EMBO J. 1984 Apr;3(4):721–731. doi: 10.1002/j.1460-2075.1984.tb01875.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Saragosti S., Cereghini S., Yaniv M. Fine structure of the regulatory region of simian virus 40 minichromosomes revealed by DNAase I digestion. J Mol Biol. 1982 Sep 15;160(2):133–146. doi: 10.1016/0022-2836(82)90171-1. [DOI] [PubMed] [Google Scholar]
  11. Saragosti S., Moyne G., Yaniv M. Absence of nucleosomes in a fraction of SV40 chromatin between the origin of replication and the region coding for the late leader RNA. Cell. 1980 May;20(1):65–73. doi: 10.1016/0092-8674(80)90235-4. [DOI] [PubMed] [Google Scholar]
  12. Stockton J. F., Miller F. D., Jorgenson K. F., Zarling D. A., Morgan A. R., Rattner J. B., van de Sande J. H. Left-handed Z-DNA regions are present in negatively supercoiled bacteriophage PM2 DNA. EMBO J. 1983;2(12):2123–2128. doi: 10.1002/j.1460-2075.1983.tb01712.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Thomae R., Beck S., Pohl F. M. Isolation of Z-DNA-containing plasmids. Proc Natl Acad Sci U S A. 1983 Sep;80(18):5550–5553. doi: 10.1073/pnas.80.18.5550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Zarling D. A., Arndt-Jovin D. J., McIntosh L. P., Robert-Nicoud M., Jovin T. M. Interactions of anti-poly[d(G-br5C)] IgG with synthetic, viral and cellular Z DNAs. J Biomol Struct Dyn. 1984 Mar;1(5):1081–1107. doi: 10.1080/07391102.1984.10507506. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. van de Sande J. H., Jovin T. M. Z* DNA, the left-handed helical form of poly[d(G-C)] in MgCl2-ethanol, is biologically active. EMBO J. 1982;1(1):115–120. doi: 10.1002/j.1460-2075.1982.tb01133.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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