Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1992 Jul 1;89(13):6055–6059. doi: 10.1073/pnas.89.13.6055

Localized torsional tension in the DNA of human cells.

M Ljungman 1, P C Hanawalt 1
PMCID: PMC49436  PMID: 1631091

Abstract

Torsional tension in DNA may be both a prerequisite for the efficient initiation of transcription and a consequence of the transcription process itself with the generation of positive torsional tension in front of the RNA polymerase and negative torsional tension behind it. To examine torsional tension in specific regions of genomic DNA in vivo, we developed an assay using photoactivated psoralen as a probe for unconstrained DNA superhelicity and x-rays as a means to relax DNA. Psoralen intercalates more readily into DNA underwound by negative torsional tension than into relaxed. DNA, and it can form interstrand DNA cross-links upon UVA irradiation. By comparing the amount of psoralen-induced DNA cross-links in cells irradiated with x-rays either before or after the psoralen treatment, we examined the topological state of the DNA in specific regions of the genome in cultured human 6A3 cells. We found that although no net torsional tension was detected in the bulk of the genome, localized tension was prominent in the DNA of two active genes. Negative torsional tension was found in the 5' end of the amplified dihydrofolate reductase gene and in a region near the 5' end of the 45S rRNA transcription unit, whereas a low level of positive torsional tension was found in a region near the 3' end of the dihydrofolate reductase gene. These results document an intragenomic heterogeneity of DNA torsional tension and lend support to the twin supercoiled domain model for transcription in the genome of intact human cells.

Full text

PDF
6058

Images in this article

6056Image
on p.6056
6056Image
on p.6056
6057Image
on p.6057

Selected References

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

  1. Ahnström G., Edvardsson K. A. Letter: Radiation-induced single-strand breaks in DNA determined by rate of alkaline strand separation and hydroxylapatite chromatography: an alternative to velocity sedimentation. Int J Radiat Biol Relat Stud Phys Chem Med. 1974 Nov;26(5):493–497. doi: 10.1080/09553007414551511. [DOI] [PubMed] [Google Scholar]
  2. Ames B. N., Gold L. S. Endogenous mutagens and the causes of aging and cancer. Mutat Res. 1991 Sep-Oct;250(1-2):3–16. doi: 10.1016/0027-5107(91)90157-j. [DOI] [PubMed] [Google Scholar]
  3. Backendorf C., Olsthoorn R., van de Putte P. Superhelical stress restrained in plasmid DNA during repair synthesis initiated by the UvrA, B and C proteins in vitro. Nucleic Acids Res. 1989 Dec 25;17(24):10337–10351. doi: 10.1093/nar/17.24.10337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bohr V. A., Smith C. A., Okumoto D. S., Hanawalt P. C. DNA repair in an active gene: removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall. Cell. 1985 Feb;40(2):359–369. doi: 10.1016/0092-8674(85)90150-3. [DOI] [PubMed] [Google Scholar]
  5. Brill S. J., Sternglanz R. Transcription-dependent DNA supercoiling in yeast DNA topoisomerase mutants. Cell. 1988 Jul 29;54(3):403–411. doi: 10.1016/0092-8674(88)90203-6. [DOI] [PubMed] [Google Scholar]
  6. Chiu S. M., Oleinick N. L., Friedman L. R., Stambrook P. J. Hypersensitivity of DNA in transcriptionally active chromatin to ionizing radiation. Biochim Biophys Acta. 1982 Oct 29;699(1):15–21. doi: 10.1016/0167-4781(82)90166-x. [DOI] [PubMed] [Google Scholar]
  7. Cimino G. D., Gamper H. B., Isaacs S. T., Hearst J. E. Psoralens as photoactive probes of nucleic acid structure and function: organic chemistry, photochemistry, and biochemistry. Annu Rev Biochem. 1985;54:1151–1193. doi: 10.1146/annurev.bi.54.070185.005443. [DOI] [PubMed] [Google Scholar]
  8. Clark D. J., Felsenfeld G. Formation of nucleosomes on positively supercoiled DNA. EMBO J. 1991 Feb;10(2):387–395. doi: 10.1002/j.1460-2075.1991.tb07960.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cook D. N., Armstrong G. A., Hearst J. E. Induction of anaerobic gene expression in Rhodobacter capsulatus is not accompanied by a local change in chromosomal supercoiling as measured by a novel assay. J Bacteriol. 1989 Sep;171(9):4836–4843. doi: 10.1128/jb.171.9.4836-4843.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Davies J. P., Thompson R. J., Mosig G. Intercalation of psoralen into DNA of plastid chromosomes decreases late during barley chloroplast development. Nucleic Acids Res. 1991 Oct 11;19(19):5219–5225. doi: 10.1093/nar/19.19.5219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dröge P., Nordheim A. Transcription-induced conformational change in a topologically closed DNA domain. Nucleic Acids Res. 1991 Jun 11;19(11):2941–2946. doi: 10.1093/nar/19.11.2941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Esposito F., Sinden R. R. DNA supercoiling and eukaryotic gene expression. Oxf Surv Eukaryot Genes. 1988;5:1–50. [PubMed] [Google Scholar]
  13. Giaever G. N., Wang J. C. Supercoiling of intracellular DNA can occur in eukaryotic cells. Cell. 1988 Dec 2;55(5):849–856. doi: 10.1016/0092-8674(88)90140-7. [DOI] [PubMed] [Google Scholar]
  14. Gruskin E. A., Lloyd R. S. Molecular analysis of plasmid DNA repair within ultraviolet-irradiated Escherichia coli. II. UvrABC-initiated excision repair and photolyase-catalyzed dimer monomerization. J Biol Chem. 1988 Sep 5;263(25):12738–12743. [PubMed] [Google Scholar]
  15. Harland R. M., Weintraub H., McKnight S. L. Transcription of DNA injected into Xenopus oocytes is influenced by template topology. Nature. 1983 Mar 3;302(5903):38–43. doi: 10.1038/302038a0. [DOI] [PubMed] [Google Scholar]
  16. Hyde J. E., Hearst J. E. Binding of psoralen derivatives to DNA and chromatin: influence of the ionic environment on dark binding and photoreactivity. Biochemistry. 1978 Apr 4;17(7):1251–1257. doi: 10.1021/bi00600a019. [DOI] [PubMed] [Google Scholar]
  17. Islas A. L., Vos J. M., Hanawalt P. C. Differential introduction and repair of psoralen photoadducts to DNA in specific human genes. Cancer Res. 1991 Jun 1;51(11):2867–2873. [PubMed] [Google Scholar]
  18. Kmiec E. B., Ryoji M., Worcel A. Gyration is required for 5S RNA transcription from a chromatin template. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1305–1309. doi: 10.1073/pnas.83.5.1305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kmiec E. B., Worcel A. The positive transcription factor of the 5S RNA gene induces a 5S DNA-specific gyration in Xenopus oocyte extracts. Cell. 1985 Jul;41(3):945–953. doi: 10.1016/s0092-8674(85)80075-1. [DOI] [PubMed] [Google Scholar]
  20. Koo H. S., Claassen L., Grossman L., Liu L. F. ATP-dependent partitioning of the DNA template into supercoiled domains by Escherichia coli UvrAB. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1212–1216. doi: 10.1073/pnas.88.4.1212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Larsen A., Weintraub H. An altered DNA conformation detected by S1 nuclease occurs at specific regions in active chick globin chromatin. Cell. 1982 Jun;29(2):609–622. doi: 10.1016/0092-8674(82)90177-5. [DOI] [PubMed] [Google Scholar]
  22. Lee M. S., Garrard W. T. Positive DNA supercoiling generates a chromatin conformation characteristic of highly active genes. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9675–9679. doi: 10.1073/pnas.88.21.9675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lee M. S., Garrard W. T. Transcription-induced nucleosome 'splitting': an underlying structure for DNase I sensitive chromatin. EMBO J. 1991 Mar;10(3):607–615. doi: 10.1002/j.1460-2075.1991.tb07988.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Leonard M. W., Patient R. K. Evidence for torsional stress in transcriptionally activated chromatin. Mol Cell Biol. 1991 Dec;11(12):6128–6138. doi: 10.1128/mcb.11.12.6128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lilley D. M. Eukaryotic genes--are they under torsional stress? Nature. 1983 Sep 22;305(5932):276–277. doi: 10.1038/305276a0. [DOI] [PubMed] [Google Scholar]
  26. Liu L. F., Wang J. C. Supercoiling of the DNA template during transcription. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7024–7027. doi: 10.1073/pnas.84.20.7024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ljungman M., Hanawalt P. C. Efficient protection against oxidative DNA damage in chromatin. Mol Carcinog. 1992;5(4):264–269. doi: 10.1002/mc.2940050406. [DOI] [PubMed] [Google Scholar]
  28. Ljungman M. Pretreatment with UV light renders the chromatin in human fibroblasts more susceptible to the DNA-damaging agents bleomycin, gamma radiation and 8-methoxypsoralen. Carcinogenesis. 1989 Mar;10(3):447–451. doi: 10.1093/carcin/10.3.447. [DOI] [PubMed] [Google Scholar]
  29. Ljungman M. The influence of chromatin structure on the frequency of radiation-induced DNA strand breaks: a study using nuclear and nucleoid monolayers. Radiat Res. 1991 Apr;126(1):58–64. [PubMed] [Google Scholar]
  30. Luchnik A. N., Hisamutdinov T. A., Georgiev G. P. Inhibition of transcription in eukaryotic cells by X-irradiation: relation to the loss of topological constraint in closed DNA loops. Nucleic Acids Res. 1988 Jun 10;16(11):5175–5190. doi: 10.1093/nar/16.11.5175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mizutani M., Ohta T., Watanabe H., Handa H., Hirose S. Negative supercoiling of DNA facilitates an interaction between transcription factor IID and the fibroin gene promoter. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):718–722. doi: 10.1073/pnas.88.3.718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mizutani M., Ura K., Hirose S. DNA superhelicity affects the formation of transcription preinitiation complex on eukaryotic genes differently. Nucleic Acids Res. 1991 Jun 11;19(11):2907–2911. doi: 10.1093/nar/19.11.2907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Morse R. H. Transcribed chromatin. Trends Biochem Sci. 1992 Jan;17(1):23–26. doi: 10.1016/0968-0004(92)90422-6. [DOI] [PubMed] [Google Scholar]
  34. Ohta T., Hirose S. Purification of a DNA supercoiling factor from the posterior silk gland of Bombyx mori. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5307–5311. doi: 10.1073/pnas.87.14.5307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Osborne B. I., Guarente L. Transcription by RNA polymerase II induces changes of DNA topology in yeast. Genes Dev. 1988 Jun;2(6):766–772. doi: 10.1101/gad.2.6.766. [DOI] [PubMed] [Google Scholar]
  36. Rahmouni A. R., Wells R. D. Direct evidence for the effect of transcription on local DNA supercoiling in vivo. J Mol Biol. 1992 Jan 5;223(1):131–144. doi: 10.1016/0022-2836(92)90721-u. [DOI] [PubMed] [Google Scholar]
  37. Ross P. M., Yu H. S. Interstrand crosslinks due to 4,5',8-trimethylpsoralen and near ultraviolet light in specific sequences of animal DNA. Effect of constitutive chromatin structure and of induced transcription. J Mol Biol. 1988 May 20;201(2):339–351. doi: 10.1016/0022-2836(88)90142-8. [DOI] [PubMed] [Google Scholar]
  38. Ryoji M., Worcel A. Chromatin assembly in Xenopus oocytes: in vivo studies. Cell. 1984 May;37(1):21–32. doi: 10.1016/0092-8674(84)90297-6. [DOI] [PubMed] [Google Scholar]
  39. Shi Y. B., Spielmann H. P., Hearst J. E. Base-catalyzed reversal of a psoralen-DNA cross-link. Biochemistry. 1988 Jul 12;27(14):5174–5178. doi: 10.1021/bi00414a034. [DOI] [PubMed] [Google Scholar]
  40. Sinden R. R., Carlson J. O., Pettijohn D. E. Torsional tension in the DNA double helix measured with trimethylpsoralen in living E. coli cells: analogous measurements in insect and human cells. Cell. 1980 Oct;21(3):773–783. doi: 10.1016/0092-8674(80)90440-7. [DOI] [PubMed] [Google Scholar]
  41. Thoma F. Structural changes in nucleosomes during transcription: strip, split or flip? Trends Genet. 1991 Jun;7(6):175–177. doi: 10.1016/0168-9525(91)90429-t. [DOI] [PubMed] [Google Scholar]
  42. Thompson R. J., Mosig G. Light affects the structure of Chlamydomonas chloroplast chromosomes. Nucleic Acids Res. 1990 May 11;18(9):2625–2631. doi: 10.1093/nar/18.9.2625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tsao Y. P., Wu H. Y., Liu L. F. Transcription-driven supercoiling of DNA: direct biochemical evidence from in vitro studies. Cell. 1989 Jan 13;56(1):111–118. doi: 10.1016/0092-8674(89)90989-6. [DOI] [PubMed] [Google Scholar]
  44. Ura K., Hirose S. Possible role of DNA topoisomerase II on transcription of the homeobox gene Hox-2.1 in F9 embryonal carcinoma cells. Nucleic Acids Res. 1991 Nov 25;19(22):6087–6092. doi: 10.1093/nar/19.22.6087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Villeponteau B., Lundell M., Martinson H. Torsional stress promotes the DNAase I sensitivity of active genes. Cell. 1984 Dec;39(3 Pt 2):469–478. doi: 10.1016/0092-8674(84)90454-9. [DOI] [PubMed] [Google Scholar]
  46. Villeponteau B., Martinson H. G. Gamma rays and bleomycin nick DNA and reverse the DNase I sensitivity of beta-globin gene chromatin in vivo. Mol Cell Biol. 1987 May;7(5):1917–1924. doi: 10.1128/mcb.7.5.1917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Vos J. M., Hanawalt P. C. Processing of psoralen adducts in an active human gene: repair and replication of DNA containing monoadducts and interstrand cross-links. Cell. 1987 Aug 28;50(5):789–799. doi: 10.1016/0092-8674(87)90337-0. [DOI] [PubMed] [Google Scholar]
  48. Wang J. C. DNA topoisomerases. Annu Rev Biochem. 1985;54:665–697. doi: 10.1146/annurev.bi.54.070185.003313. [DOI] [PubMed] [Google Scholar]
  49. Weintraub H. Assembly and propagation of repressed and depressed chromosomal states. Cell. 1985 Oct;42(3):705–711. doi: 10.1016/0092-8674(85)90267-3. [DOI] [PubMed] [Google Scholar]
  50. Weintraub H., Cheng P. F., Conrad K. Expression of transfected DNA depends on DNA topology. Cell. 1986 Jul 4;46(1):115–122. doi: 10.1016/0092-8674(86)90865-2. [DOI] [PubMed] [Google Scholar]
  51. Will C. L., Dolnick B. J. 5-Fluorouracil augmentation of dihydrofolate reductase gene transcripts containing intervening sequences in methotrexate-resistant KB cells. Mol Pharmacol. 1986 Jun;29(6):643–648. [PubMed] [Google Scholar]
  52. Wittig B., Dorbic T., Rich A. Transcription is associated with Z-DNA formation in metabolically active permeabilized mammalian cell nuclei. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2259–2263. doi: 10.1073/pnas.88.6.2259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yang J. K., Masters J. N., Attardi G. Human dihydrofolate reductase gene organization. Extensive conservation of the G + C-rich 5' non-coding sequence and strong intron size divergence from homologous mammalian genes. J Mol Biol. 1984 Jun 25;176(2):169–187. doi: 10.1016/0022-2836(84)90419-4. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES