Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Dec;178(23):6701–6705. doi: 10.1128/jb.178.23.6701-6705.1996

Interaction of Escherichia coli primase with a phage G4ori(c)-E. coli SSB complex.

W Sun 1, G N Godson 1
PMCID: PMC178564  PMID: 8955285

Abstract

We earlier reported that Escherichia coli single-stranded DNA-binding protein (SSB) bound in a fixed position to the stem-loop structure of the origin of complementary DNA strand synthesis in phage G4 (G4ori(c)), leaving stem-loop I and the adjacent 5' CTG 3', the primer RNA initiation site, as an SSB-free region (W. Sun and G. N. Godson, J. Biol. Chem. 268:8026-8039, 1993). Using a small 278-nucleotide (nt) G4ori(c) single-stranded DNA fragment that supported primer RNA synthesis, we now demonstrate by gel shift that E. coli primase can stably interact with the SSB-G4ori(c) complex. This stable interaction requires Mg2+ for specificity. At 8 mM Mg2+, primase binds to an SSB-coated 278-nt G4ori(c) fragment but not to an SSB-coated control 285-nt LacZ ss-DNA fragment. In the absence of Mg2+, primase binds to both SSB-coated fragments and gives a gel shift. T4 gene 32 protein cannot substitute for E. coli SSB in this reaction. Stable interaction of primase with naked G4ori(c). single-stranded DNA was not observed. DNase I and micrococcal nuclease footprinting, of both 5' and 3' 32P-labeled DNA, demonstrated that primase interacts with two regions of G4ori(c): one covering stem-loop I and the 3' sequence flanking stem-loop I which contains the pRNA initiation site and another located on the 5' sequence flanking stem-loop III.

Full Text

The Full Text of this article is available as a PDF (354.7 KB).

Selected References

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

  1. Arai K., Low R., Kobori J., Shlomai J., Kornberg A. Mechanism of dnaB protein action. V. Association of dnaB protein, protein n', and other repriming proteins in the primosome of DNA replication. J Biol Chem. 1981 May 25;256(10):5273–5280. [PubMed] [Google Scholar]
  2. Benz E. W., Jr, Reinberg D., Vicuna R., Hurwitz J. Initiation of DNA replication by the dnaG protein. J Biol Chem. 1980 Feb 10;255(3):1096–1106. [PubMed] [Google Scholar]
  3. Bouché J. P., Rowen L., Kornberg A. The RNA primer synthesized by primase to initiate phage G4 DNA replication. J Biol Chem. 1978 Feb 10;253(3):765–769. [PubMed] [Google Scholar]
  4. Bouché J. P., Zechel K., Kornberg A. dnaG gene product, a rifampicin-resistant RNA polymerase, initiates the conversion of a single-stranded coliphage DNA to its duplex replicative form. J Biol Chem. 1975 Aug 10;250(15):5995–6001. [PubMed] [Google Scholar]
  5. Fiddes J. C., Barrell B. G., Godson G. N. Nucleotide sequences of the separate origins of synthesis of bacteriophage G4 viral and complementary DNA strands. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1081–1085. doi: 10.1073/pnas.75.3.1081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Godson G. N. An over-expression plasmid for Escherichia coli primase. Gene. 1991 Apr;100:59–64. doi: 10.1016/0378-1119(91)90350-k. [DOI] [PubMed] [Google Scholar]
  7. Godson G. N., Barrell B. G., Staden R., Fiddes J. C. Nucleotide sequence of bacteriophage G4 DNA. Nature. 1978 Nov 16;276(5685):236–247. doi: 10.1038/276236a0. [DOI] [PubMed] [Google Scholar]
  8. Hiasa H., Sakai H., Komano T., Godson G. N. Structural features of the priming signal recognized by primase: mutational analysis of the phage G4 origin of complementary DNA strand synthesis. Nucleic Acids Res. 1990 Aug 25;18(16):4825–4831. doi: 10.1093/nar/18.16.4825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kornberg A. Enzyme systems initiating replication at the origin of the Escherichia coli chromosome. J Cell Sci Suppl. 1987;7:1–13. doi: 10.1242/jcs.1987.supplement_7.1. [DOI] [PubMed] [Google Scholar]
  10. Masai H., Arai K. Leading strand synthesis of R1 plasmid replication in vitro is primed by primase alone at a specific site downstream of oriR. J Biol Chem. 1989 May 15;264(14):8082–8090. [PubMed] [Google Scholar]
  11. Mustaev A. A., Godson G. N. Studies of the functional topography of the catalytic center of Escherichia coli primase. J Biol Chem. 1995 Jun 30;270(26):15711–15718. doi: 10.1074/jbc.270.26.15711. [DOI] [PubMed] [Google Scholar]
  12. Roth Y. F. Eucaryotic primase. Eur J Biochem. 1987 Jun 15;165(3):473–481. doi: 10.1111/j.1432-1033.1987.tb11463.x. [DOI] [PubMed] [Google Scholar]
  13. Rowen L., Kornberg A. Primase, the dnaG protein of Escherichia coli. An enzyme which starts DNA chains. J Biol Chem. 1978 Feb 10;253(3):758–764. [PubMed] [Google Scholar]
  14. Sakai H., Godson G. N. Isolation and construction of mutants of the G4 minus strand origin: analysis of their in vivo activity. Biochim Biophys Acta. 1985 Oct 3;826(1):30–37. doi: 10.1016/s0167-4781(85)80005-1. [DOI] [PubMed] [Google Scholar]
  15. Sims J., Benz E. W., Jr Initiation of DNA replication by the Escherichia coli dnaG protein: evidence that tertiary structure is involved. Proc Natl Acad Sci U S A. 1980 Feb;77(2):900–904. doi: 10.1073/pnas.77.2.900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sims J., Capon D., Dressler D. dnaG (primase)-dependent origins of DNA replication. Nucleotide sequences of the negative strand initiation sites of bacteriophages St-1, phi K, and alpha 3. J Biol Chem. 1979 Dec 25;254(24):12615–12628. [PubMed] [Google Scholar]
  17. Sims J., Dressler D. Site-specific initiation of a DNA fragment: nucleotide sequence of the bacteriophage G4 negative-strand initiation site. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3094–3098. doi: 10.1073/pnas.75.7.3094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Stayton M. M., Kornberg A. Complexes of Escherichia coli primase with the replication origin of G4 phage DNA. J Biol Chem. 1983 Nov 10;258(21):13205–13212. [PubMed] [Google Scholar]
  19. Suh W. C., Leirmo S., Record M. T., Jr Roles of Mg2+ in the mechanism of formation and dissociation of open complexes between Escherichia coli RNA polymerase and the lambda PR promoter: kinetic evidence for a second open complex requiring Mg2+. Biochemistry. 1992 Sep 1;31(34):7815–7825. doi: 10.1021/bi00149a011. [DOI] [PubMed] [Google Scholar]
  20. Sun W., Godson G. N. Binding and phasing of Escherichia coli single-stranded DNA-binding protein by the secondary structure of phage G4 origin of complementary DNA strand synthesis (G4oric). J Biol Chem. 1993 Apr 15;268(11):8026–8039. [PubMed] [Google Scholar]
  21. Swart J. R., Griep M. A. Primase from Escherichia coli primes single-stranded templates in the absence of single-stranded DNA-binding protein or other auxiliary proteins. Template sequence requirements based on the bacteriophage G4 complementary strand origin and Okazaki fragment initiation sites. J Biol Chem. 1993 Jun 15;268(17):12970–12976. [PubMed] [Google Scholar]
  22. Urlacher T. M., Griep M. A. Magnesium acetate induces a conformational change in Escherichia coli primase. Biochemistry. 1995 Dec 26;34(51):16708–16714. doi: 10.1021/bi00051a020. [DOI] [PubMed] [Google Scholar]
  23. Wickner S. DNA or RNA priming of bacteriophage G4 DNA synthesis by Escherichia coli dnaG protein. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2815–2819. doi: 10.1073/pnas.74.7.2815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wold M. S., Mallory J. B., Roberts J. D., LeBowitz J. H., McMacken R. Initiation of bacteriophage lambda DNA replication in vitro with purified lambda replication proteins. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6176–6180. doi: 10.1073/pnas.79.20.6176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Yoda K., Okazaki T. Specificity of recognition sequence for Escherichia coli primase. Mol Gen Genet. 1991 May;227(1):1–8. doi: 10.1007/BF00260698. [DOI] [PubMed] [Google Scholar]
  26. Zaychikov E., Martin E., Denissova L., Kozlov M., Markovtsov V., Kashlev M., Heumann H., Nikiforov V., Goldfarb A., Mustaev A. Mapping of catalytic residues in the RNA polymerase active center. Science. 1996 Jul 5;273(5271):107–109. doi: 10.1126/science.273.5271.107. [DOI] [PubMed] [Google Scholar]
  27. van der Ende A., Baker T. A., Ogawa T., Kornberg A. Initiation of enzymatic replication at the origin of the Escherichia coli chromosome: primase as the sole priming enzyme. Proc Natl Acad Sci U S A. 1985 Jun;82(12):3954–3958. doi: 10.1073/pnas.82.12.3954. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES