Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1993 Dec 15;12(13):4975–4983. doi: 10.1002/j.1460-2075.1993.tb06191.x

Suicidal nucleotide sequences for DNA polymerization.

G M Samadashwily 1, A Dayn 1, S M Mirkin 1
PMCID: PMC413758  PMID: 8262040

Abstract

Studying the activity of T7 DNA polymerase (Sequenase) on open circular DNAs, we observed virtually complete termination within potential triplex-forming sequences. Mutations destroying the triplex potential of the sequences prevented termination, while compensatory mutations restoring triplex potential restored it. We hypothesize that strand displacement during DNA polymerization of double-helical templates brings three DNA strands (duplex DNA downstream of the polymerase plus a displaced overhang) into close proximity, provoking triplex formation, which in turn prevents further DNA synthesis. Supporting this idea, we found that Sequenase is unable to propagate through short triple-helical stretches within single-stranded DNA templates. Thus, DNA polymerase, by inducing triplex formation at specific sequences in front of the replication fork, causes self-termination. Possible biological implications of such 'conformational suicide' are discussed. Our data also provide a novel way to target DNA polymerases at specific sequences using triplex-forming oligonucleotides.

Full text

PDF
4975

Images in this article

4977Image
on p.4977
4978Image
on p.4978
4979Image
on p.4979
4980Image
on p.4980

Selected References

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

  1. Baran N., Lapidot A., Manor H. Formation of DNA triplexes accounts for arrests of DNA synthesis at d(TC)n and d(GA)n tracts. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):507–511. doi: 10.1073/pnas.88.2.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baran N., Lapidot A., Manor H. Unusual sequence element found at the end of an amplicon. Mol Cell Biol. 1987 Jul;7(7):2636–2640. doi: 10.1128/mcb.7.7.2636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bernués J., Beltrán R., Casasnovas J. M., Azorín F. DNA-sequence and metal-ion specificity of the formation of *H-DNA. Nucleic Acids Res. 1990 Jul 25;18(14):4067–4073. doi: 10.1093/nar/18.14.4067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bernués J., Beltrán R., Casasnovas J. M., Azorín F. Structural polymorphism of homopurine--homopyrimidine sequences: the secondary DNA structure adopted by a d(GA.CT)22 sequence in the presence of zinc ions. EMBO J. 1989 Jul;8(7):2087–2094. doi: 10.1002/j.1460-2075.1989.tb03617.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Birg F., Praseuth D., Zerial A., Thuong N. T., Asseline U., Le Doan T., Hélène C. Inhibition of simian virus 40 DNA replication in CV-1 cells by an oligodeoxynucleotide covalently linked to an intercalating agent. Nucleic Acids Res. 1990 May 25;18(10):2901–2908. doi: 10.1093/nar/18.10.2901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brinton B. T., Caddle M. S., Heintz N. H. Position and orientation-dependent effects of a eukaryotic Z-triplex DNA motif on episomal DNA replication in COS-7 cells. J Biol Chem. 1991 Mar 15;266(8):5153–5161. [PubMed] [Google Scholar]
  7. Caddle M. S., Lussier R. H., Heintz N. H. Intramolecular DNA triplexes, bent DNA and DNA unwinding elements in the initiation region of an amplified dihydrofolate reductase replicon. J Mol Biol. 1990 Jan 5;211(1):19–33. doi: 10.1016/0022-2836(90)90008-A. [DOI] [PubMed] [Google Scholar]
  8. Cooney M., Czernuszewicz G., Postel E. H., Flint S. J., Hogan M. E. Site-specific oligonucleotide binding represses transcription of the human c-myc gene in vitro. Science. 1988 Jul 22;241(4864):456–459. doi: 10.1126/science.3293213. [DOI] [PubMed] [Google Scholar]
  9. Dayn A., Samadashwily G. M., Mirkin S. M. Intramolecular DNA triplexes: unusual sequence requirements and influence on DNA polymerization. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11406–11410. doi: 10.1073/pnas.89.23.11406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Duval-Valentin G., Thuong N. T., Hélène C. Specific inhibition of transcription by triple helix-forming oligonucleotides. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):504–508. doi: 10.1073/pnas.89.2.504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grigoriev M., Praseuth D., Guieysse A. L., Robin P., Thuong N. T., Hélène C., Harel-Bellan A. Inhibition of gene expression by triple helix-directed DNA cross-linking at specific sites. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3501–3505. doi: 10.1073/pnas.90.8.3501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Grigoriev M., Praseuth D., Robin P., Hemar A., Saison-Behmoaras T., Dautry-Varsat A., Thuong N. T., Hélène C., Harel-Bellan A. A triple helix-forming oligonucleotide-intercalator conjugate acts as a transcriptional repressor via inhibition of NF kappa B binding to interleukin-2 receptor alpha-regulatory sequence. J Biol Chem. 1992 Feb 15;267(5):3389–3395. [PubMed] [Google Scholar]
  13. Hanvey J. C., Klysik J., Wells R. D. Influence of DNA sequence on the formation of non-B right-handed helices in oligopurine.oligopyrimidine inserts in plasmids. J Biol Chem. 1988 May 25;263(15):7386–7396. [PubMed] [Google Scholar]
  14. Htun H., Dahlberg J. E. Single strands, triple strands, and kinks in H-DNA. Science. 1988 Sep 30;241(4874):1791–1796. doi: 10.1126/science.3175620. [DOI] [PubMed] [Google Scholar]
  15. Johnston B. H. The S1-sensitive form of d(C-T)n.d(A-G)n: chemical evidence for a three-stranded structure in plasmids. Science. 1988 Sep 30;241(4874):1800–1804. doi: 10.1126/science.2845572. [DOI] [PubMed] [Google Scholar]
  16. Kohwi Y., Kohwi-Shigematsu T. Magnesium ion-dependent triple-helix structure formed by homopurine-homopyrimidine sequences in supercoiled plasmid DNA. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3781–3785. doi: 10.1073/pnas.85.11.3781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lapidot A., Baran N., Manor H. (dT-dC)n and (dG-dA)n tracts arrest single stranded DNA replication in vitro. Nucleic Acids Res. 1989 Feb 11;17(3):883–900. doi: 10.1093/nar/17.3.883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lundquist R. C., Olivera B. M. Transient generation of displaced single-stranded DNA during nick translation. Cell. 1982 Nov;31(1):53–60. doi: 10.1016/0092-8674(82)90404-4. [DOI] [PubMed] [Google Scholar]
  19. Lyamichev V. I., Frank-Kamenetskii M. D., Soyfer V. N. Protection against UV-induced pyrimidine dimerization in DNA by triplex formation. Nature. 1990 Apr 5;344(6266):568–570. doi: 10.1038/344568a0. [DOI] [PubMed] [Google Scholar]
  20. Lyamichev V., Brow M. A., Dahlberg J. E. Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases. Science. 1993 May 7;260(5109):778–783. doi: 10.1126/science.7683443. [DOI] [PubMed] [Google Scholar]
  21. Maher L. J., 3rd, Dervan P. B., Wold B. Analysis of promoter-specific repression by triple-helical DNA complexes in a eukaryotic cell-free transcription system. Biochemistry. 1992 Jan 14;31(1):70–81. doi: 10.1021/bi00116a012. [DOI] [PubMed] [Google Scholar]
  22. Maher L. J., 3rd, Wold B., Dervan P. B. Inhibition of DNA binding proteins by oligonucleotide-directed triple helix formation. Science. 1989 Aug 18;245(4919):725–730. doi: 10.1126/science.2549631. [DOI] [PubMed] [Google Scholar]
  23. Malkov V. A., Soyfer V. N., Frank-Kamenetskii M. D. Effect of intermolecular triplex formation on the yield of cyclobutane photodimers in DNA. Nucleic Acids Res. 1992 Sep 25;20(18):4889–4895. doi: 10.1093/nar/20.18.4889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mendelsohn E., Baran N., Neer A., Manor H. Integration site of polyoma virus DNA in the inducible LPT line of polyoma-transformed rat cells. J Virol. 1982 Jan;41(1):192–209. doi: 10.1128/jvi.41.1.192-209.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Milbrandt J. D., Heintz N. H., White W. C., Rothman S. M., Hamlin J. L. Methotrexate-resistant Chinese hamster ovary cells have amplified a 135-kilobase-pair region that includes the dihydrofolate reductase gene. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6043–6047. doi: 10.1073/pnas.78.10.6043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mirkin S. M., Lyamichev V. I., Drushlyak K. N., Dobrynin V. N., Filippov S. A., Frank-Kamenetskii M. D. DNA H form requires a homopurine-homopyrimidine mirror repeat. Nature. 1987 Dec 3;330(6147):495–497. doi: 10.1038/330495a0. [DOI] [PubMed] [Google Scholar]
  27. Nelson P. S., Frye R. A., Liu E. Bifunctional oligonucleotide probes synthesized using a novel CPG support are able to detect single base pair mutations. Nucleic Acids Res. 1989 Sep 25;17(18):7187–7194. doi: 10.1093/nar/17.18.7187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Petryniak B., Lutter L. C. Topological characterization of the simian virus 40 transcription complex. Cell. 1987 Jan 30;48(2):289–295. doi: 10.1016/0092-8674(87)90432-6. [DOI] [PubMed] [Google Scholar]
  29. Postel E. H., Flint S. J., Kessler D. J., Hogan M. E. Evidence that a triplex-forming oligodeoxyribonucleotide binds to the c-myc promoter in HeLa cells, thereby reducing c-myc mRNA levels. Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):8227–8231. doi: 10.1073/pnas.88.18.8227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rao B. S., Manor H., Martin R. G. Pausing in simian virus 40 DNA replication by a sequence containing (dG-dA)27.(dT-dC)27. Nucleic Acids Res. 1988 Aug 25;16(16):8077–8094. doi: 10.1093/nar/16.16.8077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Soltis D. A., Lehman I. R. recA protein promoted DNA strand exchange. J Biol Chem. 1983 May 25;258(10):6073–6077. [PubMed] [Google Scholar]
  33. Strobel S. A., Dervan P. B. Single-site enzymatic cleavage of yeast genomic DNA mediated by triple helix formation. Nature. 1991 Mar 14;350(6314):172–174. doi: 10.1038/350172a0. [DOI] [PubMed] [Google Scholar]
  34. Strobel S. A., Doucette-Stamm L. A., Riba L., Housman D. E., Dervan P. B. Site-specific cleavage of human chromosome 4 mediated by triple-helix formation. Science. 1991 Dec 13;254(5038):1639–1642. doi: 10.1126/science.1836279. [DOI] [PubMed] [Google Scholar]
  35. Tang M. S., Htun H., Cheng Y., Dahlberg J. E. Suppression of cyclobutane and mean value of 6-4 dipyrimidines formation in triple-stranded H-DNA. Biochemistry. 1991 Jul 16;30(28):7021–7026. doi: 10.1021/bi00242a030. [DOI] [PubMed] [Google Scholar]
  36. Voloshin O. N., Mirkin S. M., Lyamichev V. I., Belotserkovskii B. P., Frank-Kamenetskii M. D. Chemical probing of homopurine-homopyrimidine mirror repeats in supercoiled DNA. Nature. 1988 Jun 2;333(6172):475–476. doi: 10.1038/333475a0. [DOI] [PubMed] [Google Scholar]
  37. Young S. L., Krawczyk S. H., Matteucci M. D., Toole J. J. Triple helix formation inhibits transcription elongation in vitro. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10023–10026. doi: 10.1073/pnas.88.22.10023. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES