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. 1982 Jun;150(3):1077–1084. doi: 10.1128/jb.150.3.1077-1084.1982

Origin and direction of mini-R1 plasmid DNA replication in cell extracts of Escherichia coli.

R Diaz, W L Staudenbauer
PMCID: PMC216325  PMID: 6281234

Abstract

Replication of the mini-R1 plasmids pKN177 and pKN182 can be carried out efficiently in cell extracts of Escherichia coli and depends on both transcription and translation. Heteroduplex and restriction analyses indicate that both plasmids are derived from the R1 copy mutant pKN104 by Is1-mediated recombination events without involving structural alterations in the replication region. To ascertain whether the in vitro replication of these miniplasmids corresponds to R1 replication in vivo, the origin and direction of replication were analyzed by electron microscopy of replicative intermediates. It was found that replication starts at a unique origin located within the RepA region at R1 coordinate at 82.4 kilobases and proceeds unidirectionally toward the IS/b sequence. The specification of the origin and the direction of in vitro replication are therefore in full agreement with the pattern observed previously for the in vivo replication of the closely related plasmids R100 and R6-5. This agreement provides additional evidence that R1 DNA synthesis in vitro employs the same replication mechanism as it does in vivo.

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Selected References

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

  1. Clerget M., Chandler M., Caro L. The structure of R1drd19: a revised physical map of the plasmid. Mol Gen Genet. 1981;181(2):183–191. doi: 10.1007/BF00268425. [DOI] [PubMed] [Google Scholar]
  2. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Danbara H., Timmis J. K., Lurz R., Timmis K. N. Plasmid replication functions: two distinct segments of plasmid R1, RepA and RepD, express incompatibility and are capable of autonomous replication. J Bacteriol. 1980 Dec;144(3):1126–1138. doi: 10.1128/jb.144.3.1126-1138.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Diaz R., Nordström K., Staudenbauer W. L. Plasmid R1 DNA replication dependent on protein synthesis in cell-free extracts of E. coli. Nature. 1981 Jan 22;289(5795):326–328. doi: 10.1038/289326a0. [DOI] [PubMed] [Google Scholar]
  5. Ely S., Staudenbauer W. L. Regulation of plasmid DNA synthesis: isolation and characterization of copy number mutants of miniR6-5 and miniF plasmids. Mol Gen Genet. 1981;181(1):29–35. doi: 10.1007/BF00339001. [DOI] [PubMed] [Google Scholar]
  6. Humphreys G. O., Willshaw G. A., Anderson E. S. A simple method for the preparation of large quantities of pure plasmid DNA. Biochim Biophys Acta. 1975 Apr 2;383(4):457–463. doi: 10.1016/0005-2787(75)90318-4. [DOI] [PubMed] [Google Scholar]
  7. Kollek R., Oertel W., Goebel W. Isolation and characterization of the minimal fragment required for autonomous replication ("basic replicon") of a copy mutant (pKN102) of the antibiotic resistance factor R1. Mol Gen Genet. 1978 Jun 1;162(1):51–57. doi: 10.1007/BF00333850. [DOI] [PubMed] [Google Scholar]
  8. Kollek R., Oertel W., Goebel W. Site-specific deletion at the replication origin of the antibiotic resistance factor R1. Mol Gen Genet. 1980 Feb;177(3):413–419. doi: 10.1007/BF00271479. [DOI] [PubMed] [Google Scholar]
  9. Mickel S., Ohtsubo E., Bauer W. Heteroduplex mapping of small plasmids derived from R-factor R12: in vivo recombination occurs at IS1 insertion sequences. Gene. 1977;2(3-4):193–210. doi: 10.1016/0378-1119(77)90017-8. [DOI] [PubMed] [Google Scholar]
  10. Molin S., Diaz R., Uhlin B. E., Nordström K. Runaway replication of plasmid R1 is not caused by loss of replication inhibitor activity of gene cop. J Bacteriol. 1980 Aug;143(2):1046–1048. doi: 10.1128/jb.143.2.1046-1048.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Molin S., Nordström K. Control of plasmid R1 replication: functions involved in replication, copy number control, incompatibility, and switch-off of replication. J Bacteriol. 1980 Jan;141(1):111–120. doi: 10.1128/jb.141.1.111-120.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Molin S., Stougaard P., Uhlin B. E., Gustafsson P., Nordström K. Clustering of genes involved in replication, copy number control, incompatibility, and stable maintenance of the resistance plasmid R1drd-19. J Bacteriol. 1979 Apr;138(1):70–79. doi: 10.1128/jb.138.1.70-79.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Morris C. F., Sinha N. K., Alberts B. M. 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):4800–4804. doi: 10.1073/pnas.72.12.4800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Oertel W., Kollek R., Beck E., Goebel W. The nucleotide sequence of a DNA fragment from the replication origin of the antibiotic resistance factor R1drd19. Mol Gen Genet. 1979 Mar 27;171(3):277–285. doi: 10.1007/BF00267582. [DOI] [PubMed] [Google Scholar]
  15. Ohtsubo E., Feingold J., Ohtsubo H., Mickel S., Bauer W. Unidirectional replication in Escherichia coli of three small plasmids derived from R factor R12. Plasmid. 1977 Nov;1(1):8–18. doi: 10.1016/0147-619x(77)90004-x. [DOI] [PubMed] [Google Scholar]
  16. Ohtsubo E., Rosenbloom M., Schrempf H., Goebel W., Rosen J. Site specific recombination involved in the generation of small plasmids. Mol Gen Genet. 1978 Feb 16;159(2):131–141. doi: 10.1007/BF00270886. [DOI] [PubMed] [Google Scholar]
  17. Ohtsubo H., Ohtsubo E. Nucleotide sequence of an insertion element, IS1. Proc Natl Acad Sci U S A. 1978 Feb;75(2):615–619. doi: 10.1073/pnas.75.2.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rosen J., Ryder T., Inokuchi H., Ohtsubo H., Ohtsubo E. Genes and sites involved in replication and incompatibility of an R100 plasmid derivative based on nucleotide sequence analysis. Mol Gen Genet. 1980;179(3):527–537. doi: 10.1007/BF00271742. [DOI] [PubMed] [Google Scholar]
  19. Sanger F., Air G. M., Barrell B. G., Brown N. L., Coulson A. R., Fiddes C. A., Hutchison C. A., Slocombe P. M., Smith M. Nucleotide sequence of bacteriophage phi X174 DNA. Nature. 1977 Feb 24;265(5596):687–695. doi: 10.1038/265687a0. [DOI] [PubMed] [Google Scholar]
  20. Staudenbauer W. L. Replication of small plasmids in extracts of Escherichia coli. Mol Gen Genet. 1976 Jun 15;145(3):273–280. doi: 10.1007/BF00325823. [DOI] [PubMed] [Google Scholar]
  21. Synenki R. M., Nordheim A., Timmis K. N. Plasmid replication functions. III. Origin and direction of replication of a "mini" plasmid derived from R6-5. Mol Gen Genet. 1979 Jan 5;168(1):27–36. doi: 10.1007/BF00267930. [DOI] [PubMed] [Google Scholar]
  22. Timmis K. N., Danbara H., Brady G., Lurz R. Inheritance functions of group IncFII transmissible antibiotic resistance plasmids. Plasmid. 1981 Jan;5(1):53–75. doi: 10.1016/0147-619x(81)90077-9. [DOI] [PubMed] [Google Scholar]
  23. Uhlin B. E., Nordström K. A runaway-replication mutant of plasmid R1drd-19: temperature-dependent loss of copy number control. Mol Gen Genet. 1978 Oct 4;165(2):167–179. doi: 10.1007/BF00269904. [DOI] [PubMed] [Google Scholar]
  24. Uhlin B. E., Nordström K. Plasmid incompatibility and control of replication: copy mutants of the R-factor R1 in Escherichia coli K-12. J Bacteriol. 1975 Nov;124(2):641–649. doi: 10.1128/jb.124.2.641-649.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Yoshikawa M. Identification and mapping of the replication genes of an R factor, R100-1, integrated into the chromosome of Escherichia coli K-12. J Bacteriol. 1974 Jun;118(3):1123–1131. doi: 10.1128/jb.118.3.1123-1131.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]

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