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. 1991 Mar 11;19(5):1129–1137. doi: 10.1093/nar/19.5.1129

A segment of a plasmid gene required for conjugal transfer encodes a site-specific, single-strand DNA endonuclease and ligase.

M K Bhattacharjee 1, R J Meyer 1
PMCID: PMC333791  PMID: 1850512

Abstract

The polypeptide encoded by a segment of a gene required for the conjugal mobilization of the broad host-range plasmid R1162 has been purified as a beta-galactosidase fusion protein. The hybrid protein binds specifically to a small, double-stranded DNA fragment containing the origin of transfer (oriT), and specifically cleaves oriT single-stranded DNA at the position cleaved during transfer. Only one of the two DNA strands is a substrate. A fraction of the digested DNA is resistant to lambda exonuclease digestion, indicating that some molecules have protein covalently attached at the 5' end. After prolonged incubation with fusion protein, some of the cleaved molecules are religated. In vivo, M13 phage DNA containing two, directly-repeated copies of oriT recombine in cells containing the fusion protein. The single-stranded viral DNA forms are the probable substrates for the protein, the cleaved DNA being subsequently religated to form recombinant molecules. Cleavage of the DNA might be the reverse reaction of the ligation that normally takes place after conjugative transfer of a single, linear plasmid DNA strand.

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Selected References

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

  1. Barlett M. M., Erickson M. J., Meyer R. J. Recombination between directly repeated origins of conjugative transfer cloned in M13 bacteriophage DNA models ligation of the transferred plasmid strand. Nucleic Acids Res. 1990 Jun 25;18(12):3579–3586. doi: 10.1093/nar/18.12.3579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brasch M. A., Meyer R. J. A 38 base-pair segment of DNA is required in cis for conjugative mobilization of broad host-range plasmid R1162. J Mol Biol. 1987 Dec 5;198(3):361–369. doi: 10.1016/0022-2836(87)90286-5. [DOI] [PubMed] [Google Scholar]
  3. Brasch M. A., Meyer R. J. Genetic organization of plasmid R1162 DNA involved in conjugative mobilization. J Bacteriol. 1986 Aug;167(2):703–710. doi: 10.1128/jb.167.2.703-710.1986. [DOI] [PMC free article] [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. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  6. Ihler G., Rupp W. D. Strand-specific transfer of donor DNA during conjugation in E. coli. Proc Natl Acad Sci U S A. 1969 May;63(1):138–143. doi: 10.1073/pnas.63.1.138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kim K., Meyer R. J. Unidirectional transfer of broad host-range plasmid R1162 during conjugative mobilization. Evidence for genetically distinct events at oriT. J Mol Biol. 1989 Aug 5;208(3):501–505. doi: 10.1016/0022-2836(89)90513-5. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Langeveld S. A., van Mansfeld A. D., de Winter J. M., Weisbeek P. J. Cleavage of single-stranded DNA by the A and A* proteins of bacteriophage phi X174. Nucleic Acids Res. 1979 Dec 20;7(8):2177–2188. doi: 10.1093/nar/7.8.2177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Little J. W. An exonuclease induced by bacteriophage lambda. II. Nature of the enzymatic reaction. J Biol Chem. 1967 Feb 25;242(4):679–686. [PubMed] [Google Scholar]
  11. Lovett M. A., Helinski D. R. Relaxation complexes of plasmid DNA and protein. II. Characterization of the proteins associated with the unrelaxed and relaxed complexes of plasmid ColE1. J Biol Chem. 1975 Nov 25;250(22):8790–8795. [PubMed] [Google Scholar]
  12. 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]
  13. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Meyer R. Site-specific recombination at oriT of plasmid R1162 in the absence of conjugative transfer. J Bacteriol. 1989 Feb;171(2):799–806. doi: 10.1128/jb.171.2.799-806.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Norrander J., Kempe T., Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983 Dec;26(1):101–106. doi: 10.1016/0378-1119(83)90040-9. [DOI] [PubMed] [Google Scholar]
  16. Scholz P., Haring V., Wittmann-Liebold B., Ashman K., Bagdasarian M., Scherzinger E. Complete nucleotide sequence and gene organization of the broad-host-range plasmid RSF1010. Gene. 1989 Feb 20;75(2):271–288. doi: 10.1016/0378-1119(89)90273-4. [DOI] [PubMed] [Google Scholar]
  17. Vapnek D., Rupp W. D. Identification of individual sex-factor DNA strands and their replication during conjugation in thermosensitive DNA mutants of Escherichia coli. J Mol Biol. 1971 Sep 28;60(3):413–424. doi: 10.1016/0022-2836(71)90178-1. [DOI] [PubMed] [Google Scholar]
  18. Willetts N., Wilkins B. Processing of plasmid DNA during bacterial conjugation. Microbiol Rev. 1984 Mar;48(1):24–41. doi: 10.1128/mr.48.1.24-41.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]

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