Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1998 Sep 1;17(17):5095–5102. doi: 10.1093/emboj/17.17.5095

Transcription through a simple DNA repeat blocks replication elongation.

M M Krasilnikova 1, G M Samadashwily 1, A S Krasilnikov 1, S M Mirkin 1
PMCID: PMC1170837  PMID: 9724645

Abstract

The influence of d(G)n.d(C)n repeats on plasmid replication in Escherichia coli cells was analyzed using electrophoretic analysis of replication intermediates. These repeats impeded the replication fork in a length- and orientation-dependent manner. Unexpectedly, the replication arrest relied primarily on the repeats' transcription. When the d(C)n sequence served as the transcriptional template, both transcription and replication were blocked. This was true for transcription driven by either bacterial or phage RNA polymerases. We hypothesize that the replication fork halts after it encounters a stalled ternary complex of the RNA polymerase, the DNA template and the r(G)n transcript. This constitutes a novel mechanism for the regulation of replication elongation. The effects of this mechanism on repeat length polymorphism and genome rearrangements are discussed.

Full Text

The Full Text of this article is available as a PDF (322.8 KB).

Selected References

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

  1. Abbotts J., SenGupta D. N., Zon G., Wilson S. H. Studies on the mechanism of Escherichia coli DNA polymerase I large fragment. Effect of template sequence and substrate variation on termination of synthesis. J Biol Chem. 1988 Oct 15;263(29):15094–15103. [PubMed] [Google Scholar]
  2. Amann E., Ochs B., Abel K. J. Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene. 1988 Sep 30;69(2):301–315. doi: 10.1016/0378-1119(88)90440-4. [DOI] [PubMed] [Google Scholar]
  3. Balbás P., Soberón X., Merino E., Zurita M., Lomeli H., Valle F., Flores N., Bolivar F. Plasmid vector pBR322 and its special-purpose derivatives--a review. Gene. 1986;50(1-3):3–40. doi: 10.1016/0378-1119(86)90307-0. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Bedinger P., Munn M., Alberts B. M. Sequence-specific pausing during in vitro DNA replication on double-stranded DNA templates. J Biol Chem. 1989 Oct 5;264(28):16880–16886. [PubMed] [Google Scholar]
  6. Bowater R. P., Jaworski A., Larson J. E., Parniewski P., Wells R. D. Transcription increases the deletion frequency of long CTG.CAG triplet repeats from plasmids in Escherichia coli. Nucleic Acids Res. 1997 Jul 15;25(14):2861–2868. doi: 10.1093/nar/25.14.2861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brewer B. J., Fangman W. L. The localization of replication origins on ARS plasmids in S. cerevisiae. Cell. 1987 Nov 6;51(3):463–471. doi: 10.1016/0092-8674(87)90642-8. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Brosius J., Dull T. J., Sleeter D. D., Noller H. F. Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. J Mol Biol. 1981 May 15;148(2):107–127. doi: 10.1016/0022-2836(81)90508-8. [DOI] [PubMed] [Google Scholar]
  10. Clewell D. B. Nature of Col E 1 plasmid replication in Escherichia coli in the presence of the chloramphenicol. J Bacteriol. 1972 May;110(2):667–676. doi: 10.1128/jb.110.2.667-676.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cox R., Mirkin S. M. Characteristic enrichment of DNA repeats in different genomes. Proc Natl Acad Sci U S A. 1997 May 13;94(10):5237–5242. doi: 10.1073/pnas.94.10.5237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Dennis P. P., Nomura M. Regulation of the expression of ribosomal protein genes in Escherichia coli. J Mol Biol. 1975 Sep 5;97(1):61–76. doi: 10.1016/s0022-2836(75)80022-2. [DOI] [PubMed] [Google Scholar]
  14. Deshpande A. M., Newlon C. S. DNA replication fork pause sites dependent on transcription. Science. 1996 May 17;272(5264):1030–1033. doi: 10.1126/science.272.5264.1030. [DOI] [PubMed] [Google Scholar]
  15. French S. Consequences of replication fork movement through transcription units in vivo. Science. 1992 Nov 20;258(5086):1362–1365. doi: 10.1126/science.1455232. [DOI] [PubMed] [Google Scholar]
  16. Friedman K. L., Brewer B. J. Analysis of replication intermediates by two-dimensional agarose gel electrophoresis. Methods Enzymol. 1995;262:613–627. doi: 10.1016/0076-6879(95)62048-6. [DOI] [PubMed] [Google Scholar]
  17. Gangloff S., Lieber M. R., Rothstein R. Transcription, topoisomerases and recombination. Experientia. 1994 Mar 15;50(3):261–269. doi: 10.1007/BF01924009. [DOI] [PubMed] [Google Scholar]
  18. Grabczyk E., Fishman M. C. A long purine-pyrimidine homopolymer acts as a transcriptional diode. J Biol Chem. 1995 Jan 27;270(4):1791–1797. doi: 10.1074/jbc.270.4.1791. [DOI] [PubMed] [Google Scholar]
  19. Hacker K. J., Alberts B. M. The rapid dissociation of the T4 DNA polymerase holoenzyme when stopped by a DNA hairpin helix. A model for polymerase release following the termination of each Okazaki fragment. J Biol Chem. 1994 Sep 30;269(39):24221–24228. [PubMed] [Google Scholar]
  20. Hill T. M. Arrest of bacterial DNA replication. Annu Rev Microbiol. 1992;46:603–633. doi: 10.1146/annurev.mi.46.100192.003131. [DOI] [PubMed] [Google Scholar]
  21. Jaworski A., Blaho J. A., Larson J. E., Shimizu M., Wells R. D. Tetracycline promoter mutations decrease non-B DNA structural transitions, negative linking differences and deletions in recombinant plasmids in Escherichia coli. J Mol Biol. 1989 Jun 5;207(3):513–526. doi: 10.1016/0022-2836(89)90461-0. [DOI] [PubMed] [Google Scholar]
  22. Kiyama R., Oishi M. In vitro transcription of a poly(dA) x poly(dT)-containing sequence is inhibited by interaction between the template and its transcripts. Nucleic Acids Res. 1996 Nov 15;24(22):4577–4583. doi: 10.1093/nar/24.22.4577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kiyama R., Oishi M. Instability of plasmid DNA maintenance caused by transcription of poly(dT)-containing sequences in Escherichia coli. Gene. 1994 Dec 2;150(1):57–61. doi: 10.1016/0378-1119(94)90857-5. [DOI] [PubMed] [Google Scholar]
  24. Kohwi Y., Malkhosyan S. R., Kohwi-Shigematsu T. Intramolecular dG.dG.dC triplex detected in Escherichia coli cells. J Mol Biol. 1992 Feb 20;223(4):817–822. doi: 10.1016/0022-2836(92)90242-c. [DOI] [PubMed] [Google Scholar]
  25. Krasilnikov A. S., Panyutin I. G., Samadashwily G. M., Cox R., Lazurkin Y. S., Mirkin S. M. Mechanisms of triplex-caused polymerization arrest. Nucleic Acids Res. 1997 Apr 1;25(7):1339–1346. doi: 10.1093/nar/25.7.1339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lalioti M. D., Scott H. S., Buresi C., Rossier C., Bottani A., Morris M. A., Malafosse A., Antonarakis S. E. Dodecamer repeat expansion in cystatin B gene in progressive myoclonus epilepsy. Nature. 1997 Apr 24;386(6627):847–851. doi: 10.1038/386847a0. [DOI] [PubMed] [Google Scholar]
  27. Liu B., Alberts B. M. Head-on collision between a DNA replication apparatus and RNA polymerase transcription complex. Science. 1995 Feb 24;267(5201):1131–1137. doi: 10.1126/science.7855590. [DOI] [PubMed] [Google Scholar]
  28. Liu B., Wong M. L., Tinker R. L., Geiduschek E. P., Alberts B. M. The DNA replication fork can pass RNA polymerase without displacing the nascent transcript. Nature. 1993 Nov 4;366(6450):33–39. doi: 10.1038/366033a0. [DOI] [PubMed] [Google Scholar]
  29. Marians K. J. Prokaryotic DNA replication. Annu Rev Biochem. 1992;61:673–719. doi: 10.1146/annurev.bi.61.070192.003325. [DOI] [PubMed] [Google Scholar]
  30. Martín-Parras L., Hernández P., Martínez-Robles M. L., Schvartzman J. B. Unidirectional replication as visualized by two-dimensional agarose gel electrophoresis. J Mol Biol. 1991 Aug 20;220(4):843–853. doi: 10.1016/0022-2836(91)90357-c. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Reaban M. E., Griffin J. A. Induction of RNA-stabilized DNA conformers by transcription of an immunoglobulin switch region. Nature. 1990 Nov 22;348(6299):342–344. doi: 10.1038/348342a0. [DOI] [PubMed] [Google Scholar]
  34. Reaban M. E., Lebowitz J., Griffin J. A. Transcription induces the formation of a stable RNA.DNA hybrid in the immunoglobulin alpha switch region. J Biol Chem. 1994 Aug 26;269(34):21850–21857. [PubMed] [Google Scholar]
  35. Rooney S. M., Moore P. D. Antiparallel, intramolecular triplex DNA stimulates homologous recombination in human cells. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2141–2144. doi: 10.1073/pnas.92.6.2141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Samadashwily G. M., Raca G., Mirkin S. M. Trinucleotide repeats affect DNA replication in vivo. Nat Genet. 1997 Nov;17(3):298–304. doi: 10.1038/ng1197-298. [DOI] [PubMed] [Google Scholar]
  37. Stanton L. W., Marcu K. B. Nucleotide sequence and properties of the murine gamma 3 immunoglobulin heavy chain gene switch region: implications for successive C gamma gene switching. Nucleic Acids Res. 1982 Oct 11;10(19):5993–6006. doi: 10.1093/nar/10.19.5993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Stavnezer J., Radcliffe G., Lin Y. C., Nietupski J., Berggren L., Sitia R., Severinson E. Immunoglobulin heavy-chain switching may be directed by prior induction of transcripts from constant-region genes. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7704–7708. doi: 10.1073/pnas.85.20.7704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  40. Uptain S. M., Kane C. M., Chamberlin M. J. Basic mechanisms of transcript elongation and its regulation. Annu Rev Biochem. 1997;66:117–172. doi: 10.1146/annurev.biochem.66.1.117. [DOI] [PubMed] [Google Scholar]
  41. Usdin K., Woodford K. J. CGG repeats associated with DNA instability and chromosome fragility form structures that block DNA synthesis in vitro. Nucleic Acids Res. 1995 Oct 25;23(20):4202–4209. doi: 10.1093/nar/23.20.4202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wahls W. P., Wallace L. J., Moore P. D. Hypervariable minisatellite DNA is a hotspot for homologous recombination in human cells. Cell. 1990 Jan 12;60(1):95–103. doi: 10.1016/0092-8674(90)90719-u. [DOI] [PubMed] [Google Scholar]
  43. Weitzmann M. N., Woodford K. J., Usdin K. The development and use of a DNA polymerase arrest assay for the evaluation of parameters affecting intrastrand tetraplex formation. J Biol Chem. 1996 Aug 23;271(34):20958–20964. doi: 10.1074/jbc.271.34.20958. [DOI] [PubMed] [Google Scholar]
  44. Woodford K. J., Howell R. M., Usdin K. A novel K(+)-dependent DNA synthesis arrest site in a commonly occurring sequence motif in eukaryotes. J Biol Chem. 1994 Oct 28;269(43):27029–27035. [PubMed] [Google Scholar]

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

RESOURCES