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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
. 1987 Dec;84(23):8345–8349. doi: 10.1073/pnas.84.23.8345

Escherichia coli replication factor Y, a component of the primosome, can act as a DNA helicase.

M S Lee 1, K J Marians 1
PMCID: PMC299539  PMID: 2825188

Abstract

The primosome is a mobile multienzyme DNA replication-priming complex that requires seven Escherichia coli proteins for assembly (the products of the dnaB, dnaC, dnaG, and dnaT genes as well as proteins n and n" and replication factor Y). It has been shown previously that the primosome, in combination with the E. coli DNA polymerase III holoenzyme, can form replication forks in vitro that move at rates similar to those measured in vivo and that the primosome and one of the components of the primosome, the DNA B protein, have DNA helicase activity. Evidence is presented here that another component of the primosome, replication factor Y, possesses DNA helicase activity as well. Factor Y helicase activity requires the presence of E. coli single-stranded DNA binding protein, Mg2+, and hydrolyzable ATP or dATP. Helicase activity is stimulated 15-fold when the enzyme is actively loaded onto single-stranded DNA through a primosome assembly site, and duplex DNA is unwound unidirectionally, 3'----5', along the DNA strand to which the protein is bound.

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Selected References

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  1. Abdel-Monem M., Dürwald H., Hoffmann-Berling H. DNA unwinding enzyme II of Escherichia coli. 2. Characterization of the DNA unwinding activity. Eur J Biochem. 1977 Sep 15;79(1):39–45. doi: 10.1111/j.1432-1033.1977.tb11781.x. [DOI] [PubMed] [Google Scholar]
  2. Abdel-Monem M., Dürwald H., Hoffmann-Berling H. Enzymic unwinding of DNA. 2. Chain separation by an ATP-dependent DNA unwinding enzyme. Eur J Biochem. 1976 Jun 1;65(2):441–449. doi: 10.1111/j.1432-1033.1976.tb10359.x. [DOI] [PubMed] [Google Scholar]
  3. Alberts B. M., Barry J., Bedinger P., Formosa T., Jongeneel C. V., Kreuzer K. N. Studies on DNA replication in the bacteriophage T4 in vitro system. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 2):655–668. doi: 10.1101/sqb.1983.047.01.077. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Arai K., Low R. L., Kornberg A. Movement and site selection for priming by the primosome in phage phi X174 DNA replication. Proc Natl Acad Sci U S A. 1981 Feb;78(2):707–711. doi: 10.1073/pnas.78.2.707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Bedinger P., Hochstrasser M., Jongeneel C. V., Alberts B. M. Properties of the T4 bacteriophage DNA replication apparatus: the T4 dda DNA helicase is required to pass a bound RNA polymerase molecule. Cell. 1983 Aug;34(1):115–123. doi: 10.1016/0092-8674(83)90141-1. [DOI] [PubMed] [Google Scholar]
  8. Boeke J. D. One and two codon insertion mutants of bacteriophage f1. Mol Gen Genet. 1981;181(3):288–291. doi: 10.1007/BF00425599. [DOI] [PubMed] [Google Scholar]
  9. Francke B., Ray D. S. Fate of parental phi X174-DNA upon infection of starved thymine-requiring host cells. Virology. 1971 Apr;44(1):168–187. doi: 10.1016/0042-6822(71)90163-2. [DOI] [PubMed] [Google Scholar]
  10. Greenbaum J. H., Marians K. J. Mutational analysis of primosome assembly sites. Evidence for alternative DNA structures. J Biol Chem. 1985 Oct 5;260(22):12266–12272. [PubMed] [Google Scholar]
  11. Greenbaum J. H., Marians K. J. The interaction of Escherichia coli replication factor Y with complementary strand origins of DNA replication. Contact points revealed by DNase footprinting and protection from methylation. J Biol Chem. 1984 Feb 25;259(4):2594–2601. [PubMed] [Google Scholar]
  12. Kreuzer K. N., Alberts B. M. Site-specific recognition of bacteriophage T4 DNA by T4 type II DNA topoisomerase and Escherichia coli DNA gyrase. J Biol Chem. 1984 Apr 25;259(8):5339–5346. [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. Liu C. C., Alberts B. M. Characterization of the DNA-dependent GTPase activity of T4 gene 41 protein, an essential component of the T4 bacteriophage DNA replication apparatus. J Biol Chem. 1981 Mar 25;256(6):2813–2820. [PubMed] [Google Scholar]
  15. Marians K. J. Enzymology of DNA in replication in prokaryotes. CRC Crit Rev Biochem. 1984;17(2):153–215. doi: 10.3109/10409238409113604. [DOI] [PubMed] [Google Scholar]
  16. Matson S. W., Tabor S., Richardson C. C. The gene 4 protein of bacteriophage T7. Characterization of helicase activity. J Biol Chem. 1983 Nov 25;258(22):14017–14024. [PubMed] [Google Scholar]
  17. 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]
  18. Model P., Zinder N. D. In vitro synthesis of bacteriophage f1 proteins. J Mol Biol. 1974 Feb 25;83(2):231–251. doi: 10.1016/0022-2836(74)90389-1. [DOI] [PubMed] [Google Scholar]
  19. Mok M., Marians K. J. Formation of rolling-circle molecules during phi X174 complementary strand DNA replication. J Biol Chem. 1987 Feb 15;262(5):2304–2309. [PubMed] [Google Scholar]
  20. Nomura N., Low R. L., Ray D. S. Selective cloning of Co1E1 DNA initiation sequences using the cloning vector M13 delta E101. Gene. 1982 Jun;18(3):239–246. doi: 10.1016/0378-1119(82)90161-5. [DOI] [PubMed] [Google Scholar]
  21. Scott J. F., Eisenberg S., Bertsch L. L., Kornberg A. A mechanism of duplex DNA replication revealed by enzymatic studies of phage phi X174: catalytic strand separation in advance of replication. Proc Natl Acad Sci U S A. 1977 Jan;74(1):193–197. doi: 10.1073/pnas.74.1.193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Shlomai J., Kornberg A. A prepriming DNA replication enzyme of Escherichia coli. I. Purification of protein n': a sequence-specific, DNA-dependent ATPase. J Biol Chem. 1980 Jul 25;255(14):6789–6793. [PubMed] [Google Scholar]
  23. Shlomai J., Kornberg A. A prepriming DNA replication enzyme of Escherichia coli. II. Actions of protein n': a sequence-specific, DNA-dependent ATPase. J Biol Chem. 1980 Jul 25;255(14):6794–6798. [PubMed] [Google Scholar]
  24. Shlomai J., Kornberg A. An Escherichia coli replication protein that recognizes a unique sequence within a hairpin region in phi X174 DNA. Proc Natl Acad Sci U S A. 1980 Feb;77(2):799–803. doi: 10.1073/pnas.77.2.799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Venkatesan M., Silver L. L., Nossal N. G. Bacteriophage T4 gene 41 protein, required for the synthesis of RNA primers, is also a DNA helicase. J Biol Chem. 1982 Oct 25;257(20):12426–12434. [PubMed] [Google Scholar]
  26. Weiner J. H., McMacken R., Kornberg A. Isolation of an intermediate which precedes dnaG RNA polymerase participation in enzymatic replication of bacteriophage phi X174 DNA. Proc Natl Acad Sci U S A. 1976 Mar;73(3):752–756. doi: 10.1073/pnas.73.3.752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wickner S., Hurwitz J. Association of phiX174 DNA-dependent ATPase activity with an Escherichia coli protein, replication factor Y, required for in vitro synthesis of phiX174 DNA. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3342–3346. doi: 10.1073/pnas.72.9.3342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Zipursky S. L., Marians K. J. Escherichia coli factor Y sites of plasmid pBR322 can function as origins of DNA replication. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6111–6115. doi: 10.1073/pnas.78.10.6111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zipursky S. L., Marians K. J. Identification of two Escherichia coli factor Y effector sites near the origins of replication of the plasmids (ColE1 and pBR322. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6521–6525. doi: 10.1073/pnas.77.11.6521. [DOI] [PMC free article] [PubMed] [Google Scholar]

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