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. 1985 Sep;4(9):2301–2307. doi: 10.1002/j.1460-2075.1985.tb03930.x

A single amino acid alteration in the initiation protein is responsible for the DNA overproduction phenotype of copy number mutants of plasmid R6K.

M Inuzuka, Y Wada
PMCID: PMC554501  PMID: 3000771

Abstract

A novel type of high copy-number (cop) mutants of a mini-R6K plasmid were isolated. The mutations were mapped in the pir gene which encodes the pi initiation protein for plasmid R6K DNA replication. They resulted in an alteration by substitution of a single amino acid: threonine to isoleucine at the 108th position for the cop41, and proline to serine at the 113th position for the cop50, of the 305 amino acid pi protein. The cop41 mutation in the pi protein was found to be trans-dominant over the wild-type allele in the copy control of plasmid R6K. Moreover, it was shown that the altered pi protein was not overproduced in maxicells carrying this mutant plasmid and had a higher affinity to the repeated sequence which is present in the pir promoter region. Most likely the mutated pi protein also interacts more efficiently with the same repeated sequences, a target of pi, in the replication origin region and increases the frequency of the initiation event per cell division.

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Selected References

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

  1. Armstrong K. A., Acosta R., Ledner E., Machida Y., Pancotto M., McCormick M., Ohtsubo H., Ohtsubo E. A 37 X 10(3) molecular weight plasmid-encoded protein is required for replication and copy number control in the plasmid pSC101 and its temperature-sensitive derivative pHS1. J Mol Biol. 1984 May 25;175(3):331–348. doi: 10.1016/0022-2836(84)90352-8. [DOI] [PubMed] [Google Scholar]
  2. Baumstark B. R., Lowery K., Scott J. R. Location by DNA sequence analysis of cop mutations affecting the number of plasmid copies of prophage P1. Mol Gen Genet. 1984;194(3):513–516. doi: 10.1007/BF00425567. [DOI] [PubMed] [Google Scholar]
  3. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  4. Casadaban M. J., Chou J., Cohen S. N. In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol. 1980 Aug;143(2):971–980. doi: 10.1128/jb.143.2.971-980.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Filutowicz M., Davis G., Greener A., Helinski D. R. Autorepressor properties of the pi-initiation protein encoded by plasmid R6K. Nucleic Acids Res. 1985 Jan 11;13(1):103–114. doi: 10.1093/nar/13.1.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Germino J., Bastia D. Interaction of the plasmid R6K-encoded replication initiator protein with its binding sites on DNA. Cell. 1983 Aug;34(1):125–134. doi: 10.1016/0092-8674(83)90142-3. [DOI] [PubMed] [Google Scholar]
  9. Germino J., Bastia D. Primary structure of the replication initiation protein of plasmid R6K. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5475–5479. doi: 10.1073/pnas.79.18.5475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hashimoto T., Sekiguchi M. Isolation of temperature-sensitive mutants of R plasmid by in vitro mutagenesis with hydroxylamine. J Bacteriol. 1976 Sep;127(3):1561–1563. doi: 10.1128/jb.127.3.1561-1563.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Inuzuka M., Helinski D. R. Replication of antibiotic resistance plasmid R6K DNA in vitro. Biochemistry. 1978 Jun 27;17(13):2567–2573. doi: 10.1021/bi00606a017. [DOI] [PubMed] [Google Scholar]
  12. Inuzuka M., Helinski D. R. Requirement of a plasmid-encoded protein for replication in vitro of plasmid R6K. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5381–5385. doi: 10.1073/pnas.75.11.5381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Inuzuka M. Plasmid-encoded initiation protein is required for activity at all three origins of plasmid R6K DNA replication in vitro. FEBS Lett. 1985 Feb 25;181(2):236–240. doi: 10.1016/0014-5793(85)80266-0. [DOI] [PubMed] [Google Scholar]
  14. Inuzuka N., Inuzuka M., Helinski D. R. Activity in vitro of three replication origins of the antibiotic resistance plasmid RSF1040. J Biol Chem. 1980 Dec 10;255(23):11071–11074. [PubMed] [Google Scholar]
  15. Inuzuka N., Inuzuka M., Helinski D. R. Activity in vitro of three replication origins of the antibiotic resistance plasmid RSF1040. J Biol Chem. 1980 Dec 10;255(23):11071–11074. [PubMed] [Google Scholar]
  16. Kahn M., Kolter R., Thomas C., Figurski D., Meyer R., Remaut E., Helinski D. R. Plasmid cloning vehicles derived from plasmids ColE1, F, R6K, and RK2. Methods Enzymol. 1979;68:268–280. doi: 10.1016/0076-6879(79)68019-9. [DOI] [PubMed] [Google Scholar]
  17. Kamio Y., Tabuchi A., Itoh Y., Katagiri H., Terawaki Y. Complete nucleotide sequence of mini-Rts1 and its copy mutant. J Bacteriol. 1984 Apr;158(1):307–312. doi: 10.1128/jb.158.1.307-312.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kelley W., Bastia D. Replication initiator protein of plasmid R6K autoregulates its own synthesis at the transcriptional step. Proc Natl Acad Sci U S A. 1985 May;82(9):2574–2578. doi: 10.1073/pnas.82.9.2574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kolter R., Helinski D. R. Construction of plasmid R6K derivatives in vitro: characterization of the R6K replication region. Plasmid. 1978 Sep;1(4):571–580. doi: 10.1016/0147-619x(78)90014-8. [DOI] [PubMed] [Google Scholar]
  20. Kolter R., Helinski D. R. Plasmid R6K DNA replication. II. Direct nucleotide sequence repeats are required for an active gamma-origin. J Mol Biol. 1982 Oct 15;161(1):45–56. doi: 10.1016/0022-2836(82)90277-7. [DOI] [PubMed] [Google Scholar]
  21. Kolter R., Inuzuka M., Helinski D. R. Trans-complementation-dependent replication of a low molecular weight origin fragment from plasmid R6K. Cell. 1978 Dec;15(4):1199–1208. doi: 10.1016/0092-8674(78)90046-6. [DOI] [PubMed] [Google Scholar]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  24. McEachern M. J., Filutowicz M., Helinski D. R. Mutations in direct repeat sequences and in a conserved sequence adjacent to the repeats result in a defective replication origin in plasmid R6K. Proc Natl Acad Sci U S A. 1985 Mar;82(5):1480–1484. doi: 10.1073/pnas.82.5.1480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nag D. K., Chattopadhyay D. J., Mandal N. C. Structure and function of the repressor of bacteriophage lambda. II. Isolation and characterization of a lambda mutant which produces repressor having higher affinity for operators. Mol Gen Genet. 1984;194(3):373–376. doi: 10.1007/BF00425547. [DOI] [PubMed] [Google Scholar]
  26. Nordström K., Molin S., Light J. Control of replication of bacterial plasmids: genetics, molecular biology, and physiology of the plasmid R1 system. Plasmid. 1984 Sep;12(2):71–90. doi: 10.1016/0147-619x(84)90054-4. [DOI] [PubMed] [Google Scholar]
  27. Pabo C. O., Sauer R. T. Protein-DNA recognition. Annu Rev Biochem. 1984;53:293–321. doi: 10.1146/annurev.bi.53.070184.001453. [DOI] [PubMed] [Google Scholar]
  28. Pheiffer B. H., Zimmerman S. B. Polymer-stimulated ligation: enhanced blunt- or cohesive-end ligation of DNA or deoxyribooligonucleotides by T4 DNA ligase in polymer solutions. Nucleic Acids Res. 1983 Nov 25;11(22):7853–7871. doi: 10.1093/nar/11.22.7853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Scott J. R. Regulation of plasmid replication. Microbiol Rev. 1984 Mar;48(1):1–23. doi: 10.1016/b978-0-12-048850-6.50006-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Seelke R. W., Kline B. C., Trawick J. D., Ritts G. D. Genetic studies of F plasmid maintenance genes involved in copy number control, incompatability, and partitioning. Plasmid. 1982 Mar;7(2):163–179. doi: 10.1016/0147-619x(82)90075-0. [DOI] [PubMed] [Google Scholar]
  31. Shafferman A., Kolter R., Stalker D., Helinski D. R. Plasmid R6K DNA replication. III. Regulatory properties of the pi initiation protein. J Mol Biol. 1982 Oct 15;161(1):57–76. doi: 10.1016/0022-2836(82)90278-9. [DOI] [PubMed] [Google Scholar]
  32. Stalker D. M., Filutowicz M., Helinski D. R. Release of initiation control by a mutational alteration in the R6K pi protein required for plasmid DNA replication. Proc Natl Acad Sci U S A. 1983 Sep;80(18):5500–5504. doi: 10.1073/pnas.80.18.5500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sutcliffe J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):77–90. doi: 10.1101/sqb.1979.043.01.013. [DOI] [PubMed] [Google Scholar]
  34. Tsurushita N., Hirano M., Shigesada K., Imai M. Isolation and characterization of rho mutants of Escherichia coli with increased transcription termination activities. Mol Gen Genet. 1984;196(3):458–464. doi: 10.1007/BF00436193. [DOI] [PubMed] [Google Scholar]
  35. Uhlin B. E., Nordström K. R plasmid gene dosage effects in Escherichia coli K-12: copy mutants of the R plasmic R1drd-19. Plasmid. 1977 Nov;1(1):1–7. doi: 10.1016/0147-619x(77)90003-8. [DOI] [PubMed] [Google Scholar]
  36. Wieslander L. A simple method to recover intact high molecular weight RNA and DNA after electrophoretic separation in low gelling temperature agarose gels. Anal Biochem. 1979 Oct 1;98(2):305–309. doi: 10.1016/0003-2697(79)90145-3. [DOI] [PubMed] [Google Scholar]
  37. Wong E. M., Muesing M. A., Polisky B. Temperature-sensitive copy number mutants of CoIE1 are located in an untranslated region of the plasmid genome. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3570–3574. doi: 10.1073/pnas.79.11.3570. [DOI] [PMC free article] [PubMed] [Google Scholar]

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