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. 1997 Sep;179(18):5795–5801. doi: 10.1128/jb.179.18.5795-5801.1997

Functional features of an ssi signal of plasmid pGKV21 in Escherichia coli.

J Y Jeong 1, G M Yoon 1, H S Seo 1, M J Cho 1, H Sakai 1, T Komano 1, J D Bahk 1
PMCID: PMC179469  PMID: 9294437

Abstract

A single-strand initiation (ssi) signal was detected on the Lactococcus lactis plasmid pGKV21 containing the replicon of pWV01 by its ability to complement the poor growth of an M13 phage derivative (M13 delta lac182) lacking the complementary-strand origin in Escherichia coli. This ssi signal was situated at the 229-nucleotide (nt) DdeI-DraI fragment and located within the 109 nt upstream of the nick site of the putative plus origin. SSI activity is orientation specific with respect to the direction of replication. We constructed an ssi signal-deleted plasmid and then examined the effects of the ssi signal on the conversion of the single-stranded replication intermediate to double-stranded plasmid DNA in E. coli. The plasmid lacking an ssi signal accumulated much more plasmid single-stranded DNA than the wild-type plasmid did. Moreover, deletion of this region caused a great reduction in plasmid copy number or plasmid maintenance. These results suggest that in E. coli, this ssi signal directs its lagging-strand synthesis as a minus origin of plasmid pGKV21. Primer RNA synthesis in vitro suggests that E. coli RNA polymerase directly recognizes the 229-nt ssi signal and synthesizes primer RNA dependent on the presence of E. coli single-stranded DNA binding (SSB) protein. This region contains two stem-loop structures, stem-loop I and stem-loop II. Deletion of stem-loop I portion results in loss of priming activity by E. coli RNA polymerase, suggesting that stem-loop I portion is essential for priming by E. coli RNA polymerase on the SSB-coated single-stranded DNA template.

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Selected References

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

  1. 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]
  2. Bahk J. D., Kioka N., Sakai H., Komano T. A runaway-replication plasmid pSY343 contains two ssi signals. Plasmid. 1988 Nov;20(3):266–270. doi: 10.1016/0147-619x(88)90033-9. [DOI] [PubMed] [Google Scholar]
  3. Bahk J. D., Sakai H., Komano T. Plasmid pACYC184 contains an ssi signal for initiation of single-strand phage DNA replication. Gene. 1988 May 15;65(1):93–99. doi: 10.1016/0378-1119(88)90420-9. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Geider K., Beck E., Schaller H. An RNA transcribed from DNA at the origin of phage fd single strand to replicative form conversion. Proc Natl Acad Sci U S A. 1978 Feb;75(2):645–649. doi: 10.1073/pnas.75.2.645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gros M. F., te Riele H., Ehrlich S. D. Rolling circle replication of single-stranded DNA plasmid pC194. EMBO J. 1987 Dec 1;6(12):3863–3869. doi: 10.1002/j.1460-2075.1987.tb02724.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gruss A. D., Ross H. F., Novick R. P. Functional analysis of a palindromic sequence required for normal replication of several staphylococcal plasmids. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2165–2169. doi: 10.1073/pnas.84.8.2165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gruss A., Ehrlich S. D. The family of highly interrelated single-stranded deoxyribonucleic acid plasmids. Microbiol Rev. 1989 Jun;53(2):231–241. doi: 10.1128/mr.53.2.231-241.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Higashitani N., Higashitani A., Roth A., Horiuchi K. SOS induction in Escherichia coli by infection with mutant filamentous phage that are defective in initiation of complementary-strand DNA synthesis. J Bacteriol. 1992 Mar;174(5):1612–1618. doi: 10.1128/jb.174.5.1612-1618.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Honda Y., Nakamura T., Tanaka K., Higashi A., Sakai H., Komano T., Bagdasarian M. DnaG-dependent priming signals can substitute for the two essential DNA initiation signals in oriV of the broad host-range plasmid RSF1010. Nucleic Acids Res. 1992 Apr 11;20(7):1733–1737. doi: 10.1093/nar/20.7.1733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Honda Y., Sakai H., Hiasa H., Tanaka K., Komano T., Bagdasarian M. Functional division and reconstruction of a plasmid replication origin: molecular dissection of the oriV of the broad-host-range plasmid RSF1010. Proc Natl Acad Sci U S A. 1991 Jan 1;88(1):179–183. doi: 10.1073/pnas.88.1.179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Honda Y., Sakai H., Komano T. Two single-strand DNA initiation signals located in the oriV region of plasmid RSF1010. Gene. 1988 Sep 7;68(2):221–228. doi: 10.1016/0378-1119(88)90024-8. [DOI] [PubMed] [Google Scholar]
  13. Imber R., Low R. L., Ray D. S. Identification of a primosome assembly site in the region of the ori 2 replication origin of the Escherichia coli mini-F plasmid. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7132–7136. doi: 10.1073/pnas.80.23.7132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kim M. H., Hines J. C., Ray D. S. Viable deletions of the M13 complementary strand origin. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6784–6788. doi: 10.1073/pnas.78.11.6784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kok J., van der Vossen J. M., Venema G. Construction of plasmid cloning vectors for lactic streptococci which also replicate in Bacillus subtilis and Escherichia coli. Appl Environ Microbiol. 1984 Oct;48(4):726–731. doi: 10.1128/aem.48.4.726-731.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Leenhouts K. J., Tolner B., Bron S., Kok J., Venema G., Seegers J. F. Nucleotide sequence and characterization of the broad-host-range lactococcal plasmid pWVO1. Plasmid. 1991 Jul;26(1):55–66. doi: 10.1016/0147-619x(91)90036-v. [DOI] [PubMed] [Google Scholar]
  17. Masai H., Nomura N., Arai K. The ABC-primosome. A novel priming system employing dnaA, dnaB, dnaC, and primase on a hairpin containing a dnaA box sequence. J Biol Chem. 1990 Sep 5;265(25):15134–15144. [PubMed] [Google Scholar]
  18. Masai H., Nomura N., Kubota Y., Arai K. Roles of phi X174 type primosome- and G4 type primase-dependent primings in initiation of lagging and leading strand syntheses of DNA replication. J Biol Chem. 1990 Sep 5;265(25):15124–15133. [PubMed] [Google Scholar]
  19. Nomura N., Low R. L., Ray D. S. Identification of ColE1 DNA sequences that direct single strand-to-double strand conversion by a phi X174 type mechanism. Proc Natl Acad Sci U S A. 1982 May;79(10):3153–3157. doi: 10.1073/pnas.79.10.3153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nomura N., Masai H., Inuzuka M., Miyazaki C., Ohtsubo E., Itoh T., Sasamoto S., Matsui M., Ishizaki R., Arai K. Identification of eleven single-strand initiation sequences (ssi) for priming of DNA replication in the F, R6K, R100 and ColE2 plasmids. Gene. 1991 Dec 1;108(1):15–22. doi: 10.1016/0378-1119(91)90482-q. [DOI] [PubMed] [Google Scholar]
  21. Ogawa T., Arai K., Okazaki T. Site selection and structure of DNA-linked RNA primers synthesized by the primosome in phage phi X174 DNA replication in vitro. J Biol Chem. 1983 Nov 10;258(21):13353–13358. [PubMed] [Google Scholar]
  22. Ray D. S., Hines J. C., Kim M. H., Imber R., Nomura N. M13 vectors for selective cloning of sequences specifying initiation of DNA synthesis on single-stranded templates. Gene. 1982 Jun;18(3):231–238. doi: 10.1016/0378-1119(82)90160-3. [DOI] [PubMed] [Google Scholar]
  23. Seegers J. F., Zhao A. C., Meijer W. J., Khan S. A., Venema G., Bron S. Structural and functional analysis of the single-strand origin of replication from the lactococcal plasmid pWV01. Mol Gen Genet. 1995 Nov 1;249(1):43–50. doi: 10.1007/BF00290234. [DOI] [PubMed] [Google Scholar]
  24. Seery L., Devine K. M. Analysis of features contributing to activity of the single-stranded origin of Bacillus plasmid pBAA1. J Bacteriol. 1993 Apr;175(7):1988–1994. doi: 10.1128/jb.175.7.1988-1994.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Shlomai J., Polder L., Arai K., Kornberg A. Replication of phi X174 dna with purified enzymes. I. Conversion of viral DNA to a supercoiled, biologically active duplex. J Biol Chem. 1981 May 25;256(10):5233–5238. [PubMed] [Google Scholar]
  27. Tanaka K., Sakai T., Honda Y., Hiasa H., Sakai H., Komano T. Plasmid Co1IB contains an ssi signal close to the replication origin. Plasmid. 1991 Mar;25(2):125–130. doi: 10.1016/0147-619x(91)90024-q. [DOI] [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. del Solar G. H., Puyet A., Espinosa M. Initiation signals for the conversion of single stranded to double stranded DNA forms in the streptococcal plasmid pLS1. Nucleic Acids Res. 1987 Jul 24;15(14):5561–5580. doi: 10.1093/nar/15.14.5561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. del Solar G., Moscoso M., Espinosa M. Rolling circle-replicating plasmids from gram-positive and gram-negative bacteria: a wall falls. Mol Microbiol. 1993 May;8(5):789–796. doi: 10.1111/j.1365-2958.1993.tb01625.x. [DOI] [PubMed] [Google Scholar]
  31. van der Vossen J. M., Kok J., Venema G. Construction of cloning, promoter-screening, and terminator-screening shuttle vectors for Bacillus subtilis and Streptococcus lactis. Appl Environ Microbiol. 1985 Aug;50(2):540–542. doi: 10.1128/aem.50.2.540-542.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]

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