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PMID: 1851552 Published · ppublish English Journal Article

Plasmid RSF1010 DNA replication in vitro promoted by purified RSF1010 RepA, RepB and RepC proteins.

Nucleic acids research ·Vol. 19 ·No. 6 ·1991-03-25 ·Pages 1203-11

Scherzinger E, Haring V, Lurz R, Otto S

Abstract

We have constructed and analyzed an in vitro system that will efficiently replicate plasmid RSF1010 and its derivatives. The system contains a partially purified extract from E.coli cells and three purified RSF1010-encoded proteins, the products of genes repA, repB (or mobA/repB), and repC. Replication in this system mimics the in vivo mechanism in that it (i) is initiated at oriV, the origin of vegetative DNA replication, (ii) proceeds in a population of plasmid molecules in both directions from this 396-base-pair origin region, and (iii) is absolutely dependent on the presence of each of the three rep gene products. In addition, we find that E.coli DNA gyrase, DnaZ protein (gamma subunit of poIIII holoenzyme) and SSB are required for in vitro plasmid synthesis. The bacterial RNA polymerase, the initiation protein DnaA, and the primosomal proteins DnaB, DnaC, DnaG and DnaT are not required. Furthermore, the replicative intermediates seen in the electron microscope suggest that replication in vitro begins with the simultaneous or non-simultaneous formation of two displacement loops that expand for a short stretch of DNA toward each other, and form a theta-type structure when the two displacing strands pass each other.

Related Genes
oriV repA repB repC
MeSH Terms
Bacterial Proteins/metabolism Coliphages/metabolism DNA Helicases DNA Polymerase II/antagonists & inhibitors,metabolism DNA Polymerase III DNA Replication DNA Topoisomerases, Type II/metabolism DNA, Bacterial/physiology,ultrastructure DNA, Superhelical/physiology,ultrastructure DNA, Viral/physiology,ultrastructure DNA-Binding Proteins DNA-Directed RNA Polymerases/antagonists & inhibitors,metabolism Electrophoresis, Polyacrylamide Gel Escherichia coli/enzymology,metabolism Microscopy, Electron Plasmids Proteins Templates, Genetic Topoisomerase II Inhibitors Trans-Activators
Chemicals
Bacterial Proteins DNA, Bacterial DNA, Superhelical DNA, Viral DNA-Binding Proteins DnaX protein, Bacteria Proteins RepB protein, Bacteria RepC protein, Staphylococcus aureus Topoisomerase II Inhibitors Trans-Activators replication initiator protein DNA-Directed RNA Polymerases DNA Polymerase II DNA Polymerase III DNA Helicases DNA Topoisomerases, Type II
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Scherzinger E
Max-Planck-Institut für Molekulare Genetik, Abteilung Schuster, Berlin, FRG.
Haring V
Lurz R
Otto S
References (39)
39 references, click to expand
  1. Aphidicolin inhibits DNA polymerase II of Escherichia coli, an alpha-like DNA polymerase.
    Nucleic Acids Res. 1990 Dec 11;18(23):7185-6 PMID: 2124684
  2. Duplex opening by dnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosome.
    Cell. 1988 Mar 11;52(5):743-55 PMID: 2830993
  3. Initiation of lagging-strand synthesis for pBR322 plasmid DNA replication in vitro is dependent on primosomal protein i encoded by dnaT.
    J Biol Chem. 1988 Oct 15;263(29):15016-23 PMID: 2844795
  4. The role of template superhelicity in the initiation of bacteriophage lambda DNA replication.
    Nucleic Acids Res. 1988 Oct 25;16(20):9611-30 PMID: 2847118
  5. Mobilization of the non-conjugative plasmid RSF1010: a genetic and DNA sequence analysis of the mobilization region.
    Mol Gen Genet. 1987 Jan;206(1):161-8 PMID: 3033438
  6. Mobilization of the non-conjugative plasmid RSF1010: a genetic analysis of its origin of transfer.
    Mol Gen Genet. 1987 Jan;206(1):154-60 PMID: 3033437
  7. Replication determinants of the broad host-range plasmid RSF1010.
    Basic Life Sci. 1985;30:243-59 PMID: 2990412
  8. Complete nucleotide sequence and gene organization of the broad-host-range plasmid RSF1010.
    Gene. 1989 Feb 20;75(2):271-88 PMID: 2653965
  9. RepB' is required in trans for the two single-strand DNA initiation signals in oriV of plasmid RSF1010.
    Gene. 1989 Aug 1;80(1):155-9 PMID: 2792769
  10. The dnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sites.
    Cell. 1984 Oct;38(3):889-900 PMID: 6091903
  11. The involvement of host replication proteins and of specific origin sequences in the in vitro replication of miniplasmid R1 DNA.
    Nucleic Acids Res. 1986 Jun 25;14(12):4865-79 PMID: 3523437
  12. Protein RepC is involved in copy number control of the broad host range plasmid RSF1010.
    Proc Natl Acad Sci U S A. 1985 Sep;82(18):6090-4 PMID: 3898079
  13. 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):15124-33 PMID: 2144283
  14. DNA topoisomerases.
    Annu Rev Biochem. 1981;50:879-910 PMID: 6267993
  15. Replication of the broad host range plasmid RSF1010 in cell-free extracts of Escherichia coli and Pseudomonas aeruginosa.
    Nucleic Acids Res. 1982 Aug 11;10(15):4687-702 PMID: 6813825
  16. The dnaZ protein, the gamma subunit of DNA polymerase III holoenzyme of Escherichia coli.
    J Biol Chem. 1980 Dec 25;255(24):11698-703 PMID: 6254978
  17. New M13 vectors for cloning.
    Methods Enzymol. 1983;101:20-78 PMID: 6310323
  18. Replication of the broad host range plasmid RSF1010: requirement for three plasmid-encoded proteins.
    Proc Natl Acad Sci U S A. 1984 Feb;81(3):654-8 PMID: 6322159
  19. The dnaC protein of Escherichia coli. Purification, physical properties and interaction with dnaB protein.
    Nucleic Acids Res. 1983 Feb 25;11(4):987-97 PMID: 6298736
  20. Electron microscopic localization of the chloroplast DNA replicative origins in Chlamydomonas reinhardii.
    Nucleic Acids Res. 1984 May 11;12(9):3843-56 PMID: 6328440
  21. Rifampicin-resistant initiation of DNA synthesis on the isolated strands of ColE plasmid DNA.
    Nucleic Acids Res. 1981 Oct 24;9(20):5215-31 PMID: 6272215
  22. A rapid boiling method for the preparation of bacterial plasmids.
    Anal Biochem. 1981 Jun;114(1):193-7 PMID: 6269464
  23. Amplification of single-strand DNA binding protein in Escherichia coli.
    Nucleic Acids Res. 1980 Jul 25;8(14):3215-27 PMID: 6449689
  24. Enzymatic replication of E. coli chromosomal origin is bidirectional.
    Nature. 1982 Apr 15;296(5858):623-7 PMID: 7040980
  25. Control of the initiation of DNA replication in Escherichia coli. I. Negative control of initiation.
    Mol Gen Genet. 1979 Jan 10;168(2):185-95 PMID: 377010
  26. A runaway-replication mutant of plasmid R1drd-19: temperature-dependent loss of copy number control.
    Mol Gen Genet. 1978 Oct 4;165(2):167-79 PMID: 366376
  27. Replication of the nonconjugative plasmid RSF1010 in Escherichia coli K-12.
    J Bacteriol. 1978 Jun;134(3):1117-22 PMID: 350840
  28. Replication of small plasmids in extracts of Escherichia coli: involvement of the dnaB and dnaC protein in the replication of early replicative intermediates.
    Mol Gen Genet. 1978 Jul 4;162(3):243-9 PMID: 355831
  29. Replication of the ampicillin resistance plasmid RSF1030 in extracts of Escherichia coli: separation of the replication cycle into early and late stages.
    Mol Gen Genet. 1977 Nov 4;156(1):27-34 PMID: 340889
  30. The electronmicroscopy of DNA.
    Annu Rev Biochem. 1974;43(0):605-19 PMID: 4137265
  31. Bacterial cell division regulation: characterization of the dnaH locus of Escherichia coli.
    J Bacteriol. 1974 Aug;119(2):443-9 PMID: 4604144
  32. Molecular nature of two nonconjugative plasmids carrying drug resistance genes.
    J Bacteriol. 1974 Feb;117(2):619-30 PMID: 4590480
  33. Pedigrees of some mutant strains of Escherichia coli K-12.
    Bacteriol Rev. 1972 Dec;36(4):525-57 PMID: 4568763
  34. phiX174 DNA-dependent DNA synthesis in vitro: requirement for P1 ban protein in dnaB mutant extracts of Escherichia coli.
    Proc Natl Acad Sci U S A. 1975 Oct;72(10):3907-11 PMID: 670
  35. Replication of a bacterial plasmid and an episome in Escherichia coli.
    Biochemistry. 1970 Jan 20;9(2):399-406 PMID: 4904868
  36. Escherichia coli mutants with temperature-sensitive synthesis of DNA.
    Mol Gen Genet. 1970;109(2):107-22 PMID: 4925091
  37. Involvement of escherichia coli dnaZ gene product in DNA elongation in vitro.
    Proc Natl Acad Sci U S A. 1976 Apr;73(4):1053-7 PMID: 772677
  38. Replication of small plasmids in extracts of Escherichia coli.
    Mol Gen Genet. 1976 Jun 15;145(3):273-80 PMID: 781516
  39. Reconstruction of bacteriophage T4 DNA replication apparatus from purified components: rolling circle replication following de novo chain initiation on a single-stranded circular DNA template.
    Proc Natl Acad Sci U S A. 1975 Dec;72(12):4800-4 PMID: 1061070
Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/19.6.1203
Published
1991-03-25
Pages
1203-11
Language
English
Region
England
NLM ID
0411011
PMCID
PMC333844
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/19.6.1203 PMC Full Text View journal details
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