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
. 1990 Apr;87(7):2481–2485. doi: 10.1073/pnas.87.7.2481

Escherichia coli Tus protein acts to arrest the progression of DNA replication forks in vitro.

T M Hill 1, K J Marians 1
PMCID: PMC53713  PMID: 2181438

Abstract

A polar DNA replication barrier is formed when the DNA-binding protein Tus forms a complex with any of the four 23-base-pair terminator (ter) sites found in the terminus region of the Escherichia coli chromosome. We have used a plasmid DNA replication system reconstituted with purified proteins in vitro to investigate the interaction of the Tus protein with the replication fork. Purified Tus protein alone is necessary and sufficient to arrest DNA replication on ColE1-type plasmid templates containing ter sites. Tus protein-catalyzed termination depends upon the orientation of the ter site in the plasmid DNA. Nucleotide resolution mapping of the terminated nascent DNA shows that leading-strand DNA synthesis arrests at the point of contact with the Tus protein, while the final lagging-strand primer sites are 50-70 nucleotides upstream. In addition, the distribution of leading-strand arrest sites changes when the composition of the proteins on the lagging-strand side of the replication fork is altered.

Full text

PDF
2481

Images in this article

2482Image
on p.2482
2483Image
on p.2483
2483Image
on p.2483
2483Image
on p.2483
2484Image
on p.2484

Selected References

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

  1. Arai K., Kornberg A. A general priming system employing only dnaB protein and primase for DNA replication. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4308–4312. doi: 10.1073/pnas.76.9.4308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arai K., Kornberg A. Unique primed start of phage phi X174 DNA replication and mobility of the primosome in a direction opposite chain synthesis. Proc Natl Acad Sci U S A. 1981 Jan;78(1):69–73. doi: 10.1073/pnas.78.1.69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. DiGate R. J., Marians K. J. Identification of a potent decatenating enzyme from Escherichia coli. J Biol Chem. 1988 Sep 15;263(26):13366–13373. [PubMed] [Google Scholar]
  4. DiGate R. J., Marians K. J. Molecular cloning and DNA sequence analysis of Escherichia coli topB, the gene encoding topoisomerase III. J Biol Chem. 1989 Oct 25;264(30):17924–17930. [PubMed] [Google Scholar]
  5. Hidaka M., Akiyama M., Horiuchi T. A consensus sequence of three DNA replication terminus sites on the E. coli chromosome is highly homologous to the terR sites of the R6K plasmid. Cell. 1988 Nov 4;55(3):467–475. doi: 10.1016/0092-8674(88)90033-5. [DOI] [PubMed] [Google Scholar]
  6. Hill T. M., Henson J. M., Kuempel P. L. The terminus region of the Escherichia coli chromosome contains two separate loci that exhibit polar inhibition of replication. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1754–1758. doi: 10.1073/pnas.84.7.1754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hill T. M., Kopp B. J., Kuempel P. L. Termination of DNA replication in Escherichia coli requires a trans-acting factor. J Bacteriol. 1988 Feb;170(2):662–668. doi: 10.1128/jb.170.2.662-668.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hill T. M., Pelletier A. J., Tecklenburg M. L., Kuempel P. L. Identification of the DNA sequence from the E. coli terminus region that halts replication forks. Cell. 1988 Nov 4;55(3):459–466. doi: 10.1016/0092-8674(88)90032-3. [DOI] [PubMed] [Google Scholar]
  9. Hill T. M., Tecklenburg M. L., Pelletier A. J., Kuempel P. L. tus, the trans-acting gene required for termination of DNA replication in Escherichia coli, encodes a DNA-binding protein. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1593–1597. doi: 10.1073/pnas.86.5.1593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Horiuchi T., Hidaka M. Core sequence of two separable terminus sites of the R6K plasmid that exhibit polar inhibition of replication is a 20 bp inverted repeat. Cell. 1988 Aug 12;54(4):515–523. doi: 10.1016/0092-8674(88)90073-6. [DOI] [PubMed] [Google Scholar]
  11. Itoh T., Tomizawa J. Purification of ribonuclease H as a factor required for initiation of in vitro Co1E1 DNA replication. Nucleic Acids Res. 1982 Oct 11;10(19):5949–5965. doi: 10.1093/nar/10.19.5949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kobayashi T., Hidaka M., Horiuchi T. Evidence of a ter specific binding protein essential for the termination reaction of DNA replication in Escherichia coli. EMBO J. 1989 Aug;8(8):2435–2441. doi: 10.1002/j.1460-2075.1989.tb08374.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. LeBowitz J. H., McMacken R. The Escherichia coli dnaB replication protein is a DNA helicase. J Biol Chem. 1986 Apr 5;261(10):4738–4748. [PubMed] [Google Scholar]
  14. Lee M. S., Marians K. J. The Escherichia coli primosome can translocate actively in either direction along a DNA strand. J Biol Chem. 1989 Aug 25;264(24):14531–14542. [PubMed] [Google Scholar]
  15. Maki H., Maki S., Kornberg A. DNA Polymerase III holoenzyme of Escherichia coli. IV. The holoenzyme is an asymmetric dimer with twin active sites. J Biol Chem. 1988 May 15;263(14):6570–6578. [PubMed] [Google Scholar]
  16. Marians K. J., Soeller W., Zipursky S. L. Maximal limits of the Escherichia coli replication factor Y effector site sequences in pBR322 DNA. J Biol Chem. 1982 May 25;257(10):5656–5662. [PubMed] [Google Scholar]
  17. McHenry C. S., Crow W. DNA polymerase III of Escherichia coli. Purification and identification of subunits. J Biol Chem. 1979 Mar 10;254(5):1748–1753. [PubMed] [Google Scholar]
  18. Minden J. S., Marians K. J. Escherichia coli topoisomerase I can segregate replicating pBR322 daughter DNA molecules in vitro. J Biol Chem. 1986 Sep 5;261(25):11906–11917. [PubMed] [Google Scholar]
  19. Minden J. S., Marians K. J. Replication of pBR322 DNA in vitro with purified proteins. Requirement for topoisomerase I in the maintenance of template specificity. J Biol Chem. 1985 Aug 5;260(16):9316–9325. [PubMed] [Google Scholar]
  20. Mok M., Marians K. J. The Escherichia coli preprimosome and DNA B helicase can form replication forks that move at the same rate. J Biol Chem. 1987 Dec 5;262(34):16644–16654. [PubMed] [Google Scholar]
  21. Pelletier A. J., Hill T. M., Kuempel P. L. Termination sites T1 and T2 from the Escherichia coli chromosome inhibit DNA replication in ColE1-derived plasmids. J Bacteriol. 1989 Mar;171(3):1739–1741. doi: 10.1128/jb.171.3.1739-1741.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sista P. R., Mukherjee S., Patel P., Khatri G. S., Bastia D. A host-encoded DNA-binding protein promotes termination of plasmid replication at a sequence-specific replication terminus. Proc Natl Acad Sci U S A. 1989 May;86(9):3026–3030. doi: 10.1073/pnas.86.9.3026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tomizawa J. I., Ohmori H., Bird R. E. Origin of replication of colicin E1 plasmid DNA. Proc Natl Acad Sci U S A. 1977 May;74(5):1865–1869. doi: 10.1073/pnas.74.5.1865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tomizawa J., Sakakibara Y., Kakefuda T. Replication of colicin E1 plasmid DNA in cell extracts. Origin and direction of replication. Proc Natl Acad Sci U S A. 1974 Jun;71(6):2260–2264. doi: 10.1073/pnas.71.6.2260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. de Massy B., Béjar S., Louarn J., Louarn J. M., Bouché J. P. Inhibition of replication forks exiting the terminus region of the Escherichia coli chromosome occurs at two loci separated by 5 min. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1759–1763. doi: 10.1073/pnas.84.7.1759. [DOI] [PMC free article] [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