Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1995 Apr 25;23(8):1292–1299. doi: 10.1093/nar/23.8.1292

Formation of DNA triple helices inhibits DNA unwinding by the SV40 large T-antigen helicase.

M Peleg 1, V Kopel 1, J A Borowiec 1, H Manor 1
PMCID: PMC306852  PMID: 7753619

Abstract

Previous studies have indicated that d(TC)n.d(GA)n microsatellites may serve as arrest signals for mammalian DNA replication through the ability of such sequences to form DNA triple helices and thereby inhibit replication enzymes. To further test this hypothesis, we examined the ability of d(TC)i.d(GA)i.d(TC)i triplexes to inhibit DNA unwinding in vitro by a model eukaryotic DNA helicase, the SV40 large T-antigen. DNA substrates that were able to form triplexes, and non-triplex-forming control substrates, were tested. We found that the presence of DNA triplexes, as assayed by endonuclease S1 and osmium tetroxide footprinting, significantly inhibited DNA unwinding by T-antigen. Strong inhibition was observed not only at acidic pH values, in which the triplexes were most stable, but also at physiological pH values in the range 6.9-7.2. Little or no inhibition was detected at pH 8.7. Based on these results, and on previous studies of DNA polymerases, we suggest that DNA triplexes may form in vivo and cause replication arrest through a dual inhibition of duplex unwinding by DNA helicases and of nascent strand synthesis by DNA polymerases. DNA triplexes also have the potential to inhibit recombination and repair processes in which helicases and polymerases are involved.

Full text

PDF
1292

Images in this article

1294Image
on p.1294
1295Image
on p.1295
1295Image
on p.1295
1297Image
on p.1297
1298Image
on p.1298

Selected References

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

  1. Aharoni A., Baran N., Manor H. Characterization of a multisubunit human protein which selectively binds single stranded d(GA)n and d(GT)n sequence repeats in DNA. Nucleic Acids Res. 1993 Nov 11;21(22):5221–5228. doi: 10.1093/nar/21.22.5221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Baran N., Neer A., Manor H. "Onion skin" replication of integrated polyoma virus DNA and flanking sequences in polyoma-transformed rat cells: termination within a specific cellular DNA segment. Proc Natl Acad Sci U S A. 1983 Jan;80(1):105–109. doi: 10.1073/pnas.80.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bergemann A. D., Johnson E. M. The HeLa Pur factor binds single-stranded DNA at a specific element conserved in gene flanking regions and origins of DNA replication. Mol Cell Biol. 1992 Mar;12(3):1257–1265. doi: 10.1128/mcb.12.3.1257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Birnboim H. C., Sederoff R. R., Paterson M. C. Distribution of polypyrimidine . polypurine segments in DNA from diverse organisms. Eur J Biochem. 1979 Jul;98(1):301–307. doi: 10.1111/j.1432-1033.1979.tb13189.x. [DOI] [PubMed] [Google Scholar]
  8. Borowiec J. A., Dean F. B., Bullock P. A., Hurwitz J. Binding and unwinding--how T antigen engages the SV40 origin of DNA replication. Cell. 1990 Jan 26;60(2):181–184. doi: 10.1016/0092-8674(90)90730-3. [DOI] [PubMed] [Google Scholar]
  9. Borowiec J. A., Dean F. B., Hurwitz J. Differential induction of structural changes in the simian virus 40 origin of replication by T antigen. J Virol. 1991 Mar;65(3):1228–1235. doi: 10.1128/jvi.65.3.1228-1235.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Cheng H. L., Blattner F. R., Fitzmaurice L., Mushinski J. F., Tucker P. W. Structure of genes for membrane and secreted murine IgD heavy chains. Nature. 1982 Apr 1;296(5856):410–415. doi: 10.1038/296410a0. [DOI] [PubMed] [Google Scholar]
  12. Crabtree G. R., Kant J. A. Organization of the rat gamma-fibrinogen gene: alternative mRNA splice patterns produce the gamma A and gamma B (gamma ') chains of fibrinogen. Cell. 1982 Nov;31(1):159–166. doi: 10.1016/0092-8674(82)90415-9. [DOI] [PubMed] [Google Scholar]
  13. Dolinnaya N. G., Fresco J. R. Single-stranded nucleic acid helical secondary structure stabilized by ionic bonds: d(A(+)-G)10. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9242–9246. doi: 10.1073/pnas.89.19.9242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fanning E., Knippers R. Structure and function of simian virus 40 large tumor antigen. Annu Rev Biochem. 1992;61:55–85. doi: 10.1146/annurev.bi.61.070192.000415. [DOI] [PubMed] [Google Scholar]
  15. François J. C., Saison-Behmoaras T., Hélène C. Sequence-specific recognition of the major groove of DNA by oligodeoxynucleotides via triple helix formation. Footprinting studies. Nucleic Acids Res. 1988 Dec 23;16(24):11431–11440. doi: 10.1093/nar/16.24.11431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Geider K., Hoffmann-Berling H. Proteins controlling the helical structure of DNA. Annu Rev Biochem. 1981;50:233–260. doi: 10.1146/annurev.bi.50.070181.001313. [DOI] [PubMed] [Google Scholar]
  17. Hanvey J. C., Shimizu M., Wells R. D. Intramolecular DNA triplexes in supercoiled plasmids. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6292–6296. doi: 10.1073/pnas.85.17.6292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hentschel C. C. Homocopolymer sequences in the spacer of a sea urchin histone gene repeat are sensitive to S1 nuclease. Nature. 1982 Feb 25;295(5851):714–716. doi: 10.1038/295714a0. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Htun H., Dahlberg J. E. Topology and formation of triple-stranded H-DNA. Science. 1989 Mar 24;243(4898):1571–1576. doi: 10.1126/science.2648571. [DOI] [PubMed] [Google Scholar]
  21. Htun H., Lund E., Dahlberg J. E. Human U1 RNA genes contain an unusually sensitive nuclease S1 cleavage site within the conserved 3' flanking region. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7288–7292. doi: 10.1073/pnas.81.23.7288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Khatri G. S., MacAllister T., Sista P. R., Bastia D. The replication terminator protein of E. coli is a DNA sequence-specific contra-helicase. Cell. 1989 Nov 17;59(4):667–674. doi: 10.1016/0092-8674(89)90012-3. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Kubinski H., Opara-Kubinska Z., Szybalski W. Patterns of interaction between polyribonucleotides and individual DNA strands derived from several vertebrates, bacteria and bacteriophages. J Mol Biol. 1966 Sep;20(2):313–329. doi: 10.1016/0022-2836(66)90067-2. [DOI] [PubMed] [Google Scholar]
  26. Kuempel P. L., Pelletier A. J., Hill T. M. Tus and the terminators: the arrest of replication in prokaryotes. Cell. 1989 Nov 17;59(4):581–583. doi: 10.1016/0092-8674(89)90001-9. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Lee E. H., Kornberg A., Hidaka M., Kobayashi T., Horiuchi T. Escherichia coli replication termination protein impedes the action of helicases. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9104–9108. doi: 10.1073/pnas.86.23.9104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Little R. D., Platt T. H., Schildkraut C. L. Initiation and termination of DNA replication in human rRNA genes. Mol Cell Biol. 1993 Oct;13(10):6600–6613. doi: 10.1128/mcb.13.10.6600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lyamichev V. I., Mirkin S. M., Frank-Kamenetskii M. D. Structures of homopurine-homopyrimidine tract in superhelical DNA. J Biomol Struct Dyn. 1986 Feb;3(4):667–669. doi: 10.1080/07391102.1986.10508454. [DOI] [PubMed] [Google Scholar]
  31. Malkov V. A., Voloshin O. N., Soyfer V. N., Frank-Kamenetskii M. D. Cation and sequence effects on stability of intermolecular pyrimidine-purine-purine triplex. Nucleic Acids Res. 1993 Feb 11;21(3):585–591. doi: 10.1093/nar/21.3.585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Matson S. W., Bean D. W., George J. W. DNA helicases: enzymes with essential roles in all aspects of DNA metabolism. Bioessays. 1994 Jan;16(1):13–22. doi: 10.1002/bies.950160103. [DOI] [PubMed] [Google Scholar]
  33. Moser H. E., Dervan P. B. Sequence-specific cleavage of double helical DNA by triple helix formation. Science. 1987 Oct 30;238(4827):645–650. doi: 10.1126/science.3118463. [DOI] [PubMed] [Google Scholar]
  34. Palecek E. Probing DNA structure with osmium tetroxide complexes in vitro. Methods Enzymol. 1992;212:139–155. doi: 10.1016/0076-6879(92)12010-n. [DOI] [PubMed] [Google Scholar]
  35. Radhakrishnan I., de los Santos C., Patel D. J. Nuclear magnetic resonance structural studies of intramolecular purine.purine.pyrimidine DNA triplexes in solution. Base triple pairing alignments and strand direction. J Mol Biol. 1991 Oct 20;221(4):1403–1418. [PubMed] [Google Scholar]
  36. Rajagopal P., Feigon J. NMR studies of triple-strand formation from the homopurine-homopyrimidine deoxyribonucleotides d(GA)4 and d(TC)4. Biochemistry. 1989 Sep 19;28(19):7859–7870. doi: 10.1021/bi00445a048. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Rao B. S. Pausing of simian virus 40 DNA replication fork movement in vivo by (dG-dA)n.(dT-dC)n tracts. Gene. 1994 Mar 25;140(2):233–237. doi: 10.1016/0378-1119(94)90549-5. [DOI] [PubMed] [Google Scholar]
  39. Samadashwily G. M., Dayn A., Mirkin S. M. Suicidal nucleotide sequences for DNA polymerization. EMBO J. 1993 Dec 15;12(13):4975–4983. doi: 10.1002/j.1460-2075.1993.tb06191.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Scheffner M., Knippers R., Stahl H. Simian-virus-40 large-T-antigen-catalyzed DNA and RNA unwinding reactions. Eur J Biochem. 1991 Jan 1;195(1):49–54. doi: 10.1111/j.1432-1033.1991.tb15674.x. [DOI] [PubMed] [Google Scholar]
  41. SenGupta D. J., Borowiec J. A. Strand-specific recognition of a synthetic DNA replication fork by the SV40 large tumor antigen. Science. 1992 Jun 19;256(5064):1656–1661. doi: 10.1126/science.256.5064.1656. [DOI] [PubMed] [Google Scholar]
  42. Singleton S. F., Dervan P. B. Influence of pH on the equilibrium association constants for oligodeoxyribonucleotide-directed triple helix formation at single DNA sites. Biochemistry. 1992 Nov 17;31(45):10995–11003. doi: 10.1021/bi00160a008. [DOI] [PubMed] [Google Scholar]
  43. Stahl H., Dröge P., Knippers R. DNA helicase activity of SV40 large tumor antigen. EMBO J. 1986 Aug;5(8):1939–1944. doi: 10.1002/j.1460-2075.1986.tb04447.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Stahl H., Knippers R. The simian virus 40 large tumor antigen. Biochim Biophys Acta. 1987 Oct 9;910(1):1–10. doi: 10.1016/0167-4781(87)90088-1. [DOI] [PubMed] [Google Scholar]
  45. Sures I., Lowry J., Kedes L. H. The DNA sequence of sea urchin (S. purpuratus) H2A, H2B and H3 histone coding and spacer regions. Cell. 1978 Nov;15(3):1033–1044. doi: 10.1016/0092-8674(78)90287-8. [DOI] [PubMed] [Google Scholar]
  46. Thömmes P., Hübscher U. Eukaryotic DNA helicases: essential enzymes for DNA transactions. Chromosoma. 1992 Jun;101(8):467–473. doi: 10.1007/BF00352468. [DOI] [PubMed] [Google Scholar]
  47. Vojtísková M., Mirkin S., Lyamichev V., Voloshin O., Frank-Kamenetskii M., Palecek E. Chemical probing of the homopurine.homopyrimidine tract in supercoiled DNA at single-nucleotide resolution. FEBS Lett. 1988 Jul 18;234(2):295–299. doi: 10.1016/0014-5793(88)80102-9. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES