• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. Dec 1995; 177(24): 7041–7049.
PMCID: PMC177580

In vitro recognition of the replication origin of pLS1 and of plasmids of the pLS1 family by the RepB initiator protein.

Abstract

Rolling-circle replication of plasmid pLS1 is initiated by the plasmid-encoded RepB protein, which has nicking-closing (site-specific DNA strand transferase) enzymatic activity. The leading-strand origin of pLS1 contains two regions, (i) the RepB-binding site, constituted by three directly repeated sequences (iterons or the bind region), and (ii) the sequence where RepB introduces the nick to initiate replication (the nic region). A series of plasmids, belonging to the pLS1 family, show features similar to those of pLS1 and have DNA sequences homologous to the pLS1 nic region. In addition, they all share homologies at the level of their Rep proteins. However, the bind regions of these plasmids are, in general, not conserved. We tested the substrate specificity of purified RepB of pLS1. The RepB protein has a temperature-dependent nicking-closing action on supercoiled pLS1, as well as on recombinant plasmid DNAs harboring the pLS1 nic region. The DNA strand transferase activity of pLS1-encoded RepB was also assayed on two plasmids of the pLS1 family, namely, pE194 and pFX2. DNAs from both plasmids were relaxed by RepB, provided they had a proper degree of supercoiling; i.e., it was necessary to modulate the supercoiling of pE194 DNA to achieve RepB-mediated DNA relaxation. Single-stranded oligonucleotides containing the nic regions of various plasmids belonging to the pLS1 family, including those of pE194 and pFX2, were substrates for RepB. In vitro, the RepB protein does not need to bind to the iterons for its nicking-closing activity.

Full Text

The Full Text of this article is available as a PDF (440K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Ballester S, Alonso JC, López P, Espinosa M. Comparative expression of the pC194 cat gene in Streptococcus pneumoniae, Bacillus subtilis and Escherichia coli. Gene. 1990 Jan 31;86(1):71–79. [PubMed]
  • Bates EE, Gilbert HJ. Characterization of a cryptic plasmid from Lactobacillus plantarum. Gene. 1989 Dec 21;85(1):253–258. [PubMed]
  • Bergemann AD, Whitley JC, Finch LR. Homology of mycoplasma plasmid pADB201 and staphylococcal plasmid pE194. J Bacteriol. 1989 Jan;171(1):593–595. [PMC free article] [PubMed]
  • Bowater R, Aboul-ela F, Lilley DM. Large-scale stable opening of supercoiled DNA in response to temperature and supercoiling in (A + T)-rich regions that promote low-salt cruciform extrusion. Biochemistry. 1991 Dec 10;30(49):11495–11506. [PubMed]
  • Coffey A, Harrington A, Kearney K, Daly C, Fitzgerald G. Nucleotide sequence and structural organization of the small, broad-host-range plasmid pCI411 from Leuconostoc lactis 533. Microbiology. 1994 Sep;140(Pt 9):2263–2269. [PubMed]
  • de la Campa AG, del Solar GH, Espinosa M. Initiation of replication of plasmid pLS1. The initiator protein RepB acts on two distant DNA regions. J Mol Biol. 1990 May 20;213(2):247–262. [PubMed]
  • del Solar GH, de al Campa AG, Pérez-Martín J, Choli T, Espinosa M. Purification and characterization of RepA, a protein involved in the copy number control of plasmid pLS1. Nucleic Acids Res. 1989 Apr 11;17(7):2405–2420. [PMC free article] [PubMed]
  • del Solar G, Diaz R, Espinosa M. Replication of the streptococcal plasmid pMV158 and derivatives in cell-free extracts of Escherichia coli. Mol Gen Genet. 1987 Mar;206(3):428–435. [PubMed]
  • del Solar G, Espinosa M. The copy number of plasmid pLS1 is regulated by two trans-acting plasmid products: the antisense RNA II and the repressor protein, RepA. Mol Microbiol. 1992 Jan;6(1):83–94. [PubMed]
  • 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. [PubMed]
  • del Solar G, Moscoso M, Espinosa M. In vivo definition of the functional origin of replication (ori(+)) of the promiscuous plasmid pLS1. Mol Gen Genet. 1993 Feb;237(1-2):65–72. [PubMed]
  • Dempsey LA, Birch P, Khan SA. Six amino acids determine the sequence-specific DNA binding and replication specificity of the initiator proteins of the pT181 family. J Biol Chem. 1992 Dec 5;267(34):24538–24543. [PubMed]
  • Gruss A, Ehrlich SD. The family of highly interrelated single-stranded deoxyribonucleic acid plasmids. Microbiol Rev. 1989 Jun;53(2):231–241. [PMC free article] [PubMed]
  • Horinouchi S, Weisblum B. Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibodies. J Bacteriol. 1982 May;150(2):804–814. [PMC free article] [PubMed]
  • Kleanthous H, Clayton CL, Tabaqchali S. Characterization of a plasmid from Helicobacter pylori encoding a replication protein common to plasmids in gram-positive bacteria. Mol Microbiol. 1991 Oct;5(10):2377–2389. [PubMed]
  • Koepsel RR, Khan SA. Cleavage of single-stranded DNA by plasmid pT181-encoded RepC protein. Nucleic Acids Res. 1987 May 26;15(10):4085–4097. [PMC free article] [PubMed]
  • Kramer MG, del Solar G, Espinosa M. Lagging-strand origins of the promiscuous plasmid pMV158: physical and functional characterization. Microbiology. 1995 Mar;141(Pt 3):655–662. [PubMed]
  • Lacks SA, Lopez P, Greenberg B, Espinosa M. Identification and analysis of genes for tetracycline resistance and replication functions in the broad-host-range plasmid pLS1. J Mol Biol. 1986 Dec 20;192(4):753–765. [PubMed]
  • Moscoso M, del Solar G, Espinosa M. Specific nicking-closing activity of the initiator of replication protein RepB of plasmid pMV158 on supercoiled or single-stranded DNA. J Biol Chem. 1995 Feb 24;270(8):3772–3779. [PubMed]
  • Noirot P, Bargonetti J, Novick RP. Initiation of rolling-circle replication in pT181 plasmid: initiator protein enhances cruciform extrusion at the origin. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8560–8564. [PMC free article] [PubMed]
  • Noirot-Gros MF, Bidnenko V, Ehrlich SD. Active site of the replication protein of the rolling circle plasmid pC194. EMBO J. 1994 Sep 15;13(18):4412–4420. [PMC free article] [PubMed]
  • Novick RP. Staphylococcal plasmids and their replication. Annu Rev Microbiol. 1989;43:537–565. [PubMed]
  • Priebe SD, Lacks SA. Region of the streptococcal plasmid pMV158 required for conjugative mobilization. J Bacteriol. 1989 Sep;171(9):4778–4784. [PMC free article] [PubMed]
  • Projan SJ, Novick R. Comparative analysis of five related Staphylococcal plasmids. Plasmid. 1988 May;19(3):203–221. [PubMed]
  • Puyet A, del Solar GH, Espinosa M. Identification of the origin and direction of replication of the broad-host-range plasmid pLS1. Nucleic Acids Res. 1988 Jan 11;16(1):115–133. [PMC free article] [PubMed]
  • Singleton CK, Wells RD. The facile generation of covalently closed, circular DNAs with defined negative superhelical densities. Anal Biochem. 1982 May 15;122(2):253–257. [PubMed]
  • Sozhamannan S, Dabert P, Moretto V, Ehrlich SD, Gruss A. Plus-origin mapping of single-stranded DNA plasmid pE194 and nick site homologies with other plasmids. J Bacteriol. 1990 Aug;172(8):4543–4548. [PMC free article] [PubMed]
  • Thomas CD, Balson DF, Shaw WV. In vitro studies of the initiation of staphylococcal plasmid replication. Specificity of RepD for its origin (oriD) and characterization of the Rep-ori tyrosyl ester intermediate. J Biol Chem. 1990 Apr 5;265(10):5519–5530. [PubMed]
  • Villafane R, Bechhofer DH, Narayanan CS, Dubnau D. Replication control genes of plasmid pE194. J Bacteriol. 1987 Oct;169(10):4822–4829. [PMC free article] [PubMed]
  • Wang PZ, Projan SJ, Henriquez V, Novick RP. Specificity of origin recognition by replication initiator protein in plasmids of the pT181 family is determined by a six amino acid residue element. J Mol Biol. 1992 Jan 5;223(1):145–158. [PubMed]
  • Wang PZ, Projan SJ, Henriquez V, Novick RP. Origin recognition specificity in pT181 plasmids is determined by a functionally asymmetric palindromic DNA element. EMBO J. 1993 Jan;12(1):45–52. [PMC free article] [PubMed]
  • Xu FF, Pearce LE, Yu PL. Genetic analysis of a lactococcal plasmid replicon. Mol Gen Genet. 1991 May;227(1):33–39. [PubMed]
  • Zock JM, Birch P, Khan SA. Specificity of RepC protein in plasmid pT181 DNA replication. J Biol Chem. 1990 Feb 25;265(6):3484–3488. [PubMed]

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

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...