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. 1988 Dec 1;7(12):3991–3996. doi: 10.1002/j.1460-2075.1988.tb03287.x

Enhancer-independent mutants of the Cin recombinase have a relaxed topological specificity.

P Haffter 1, T A Bickle 1
PMCID: PMC455005  PMID: 3208759

Abstract

Cin is a member of the hin family of complementing site-specific recombinases which regulate the alternate expression of genes by inverting DNA segments. Common characteristics of this family of recombination systems are the requirement for an enhancer-like element in cis and the specificity for inversely oriented recombination sites on the same DNA molecule. We have isolated two mutants of the Cin recombinase which will efficiently recombine a substrate lacking the enhancer. In addition, these mutant proteins also catalyse efficient recombination between sites in direct orientation or on different DNA molecules. Both mutations are due to single amino acid substitutions at different positions in the protein and the two mutants have slightly different phenotypes. The finding that the loss of enhancer dependence is coupled to a change in topological specificity leads us to conclude that the enhancer determines the specificity of the system for DNA inversion.

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Selected References

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

  1. Benjamin H. W., Cozzarelli N. R. Isolation and characterization of the Tn3 resolvase synaptic intermediate. EMBO J. 1988 Jun;7(6):1897–1905. doi: 10.1002/j.1460-2075.1988.tb03023.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boocock M. R., Brown J. L., Sherratt D. J. Structural and catalytic properties of specific complexes between Tn3 resolvase and the recombination site res. Biochem Soc Trans. 1986 Apr;14(2):214–216. doi: 10.1042/bst0140214a. [DOI] [PubMed] [Google Scholar]
  3. Bruist M. F., Glasgow A. C., Johnson R. C., Simon M. I. Fis binding to the recombinational enhancer of the Hin DNA inversion system. Genes Dev. 1987 Oct;1(8):762–772. doi: 10.1101/gad.1.8.762. [DOI] [PubMed] [Google Scholar]
  4. Bruist M. F., Simon M. I. Phase variation and the Hin protein: in vivo activity measurements, protein overproduction, and purification. J Bacteriol. 1984 Jul;159(1):71–79. doi: 10.1128/jb.159.1.71-79.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carter P., Bedouelle H., Winter G. Improved oligonucleotide site-directed mutagenesis using M13 vectors. Nucleic Acids Res. 1985 Jun 25;13(12):4431–4443. doi: 10.1093/nar/13.12.4431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Grindley N. D., Reed R. R. Transpositional recombination in prokaryotes. Annu Rev Biochem. 1985;54:863–896. doi: 10.1146/annurev.bi.54.070185.004243. [DOI] [PubMed] [Google Scholar]
  7. Haffter P., Bickle T. A. Purification and DNA-binding properties of FIS and Cin, two proteins required for the bacteriophage P1 site-specific recombination system, cin. J Mol Biol. 1987 Dec 20;198(4):579–587. doi: 10.1016/0022-2836(87)90201-4. [DOI] [PubMed] [Google Scholar]
  8. Hattori M., Sakaki Y. Dideoxy sequencing method using denatured plasmid templates. Anal Biochem. 1986 Feb 1;152(2):232–238. doi: 10.1016/0003-2697(86)90403-3. [DOI] [PubMed] [Google Scholar]
  9. Hiestand-Nauer R., Iida S. Sequence of the site-specific recombinase gene cin and of its substrates serving in the inversion of the C segment of bacteriophage P1. EMBO J. 1983;2(10):1733–1740. doi: 10.1002/j.1460-2075.1983.tb01650.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Huber H. E., Iida S., Arber W., Bickle T. A. Site-specific DNA inversion is enhanced by a DNA sequence element in cis. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3776–3780. doi: 10.1073/pnas.82.11.3776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Iida S. Bacteriophage P1 carries two related sets of genes determining its host range in the invertible C segment of its genome. Virology. 1984 Apr 30;134(2):421–434. doi: 10.1016/0042-6822(84)90309-x. [DOI] [PubMed] [Google Scholar]
  12. Iida S., Meyer J., Kennedy K. E., Arber W. A site-specific, conservative recombination system carried by bacteriophage P1. Mapping the recombinase gene cin and the cross-over sites cix for the inversion of the C segment. EMBO J. 1982;1(11):1445–1453. doi: 10.1002/j.1460-2075.1982.tb01336.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Johnson R. C., Glasgow A. C., Simon M. I. Spatial relationship of the Fis binding sites for Hin recombinational enhancer activity. Nature. 1987 Oct 1;329(6138):462–465. doi: 10.1038/329462a0. [DOI] [PubMed] [Google Scholar]
  14. Johnson R. C., Simon M. I. Hin-mediated site-specific recombination requires two 26 bp recombination sites and a 60 bp recombinational enhancer. Cell. 1985 Jul;41(3):781–791. doi: 10.1016/s0092-8674(85)80059-3. [DOI] [PubMed] [Google Scholar]
  15. Kahmann R., Rudt F., Koch C., Mertens G. G inversion in bacteriophage Mu DNA is stimulated by a site within the invertase gene and a host factor. Cell. 1985 Jul;41(3):771–780. doi: 10.1016/s0092-8674(85)80058-1. [DOI] [PubMed] [Google Scholar]
  16. Kamp D., Chow L. T., Broker T. R., Kwoh D., Zipser D., Kahmann R. Site-specific recombination in phage mu. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 2):1159–1167. doi: 10.1101/sqb.1979.043.01.131. [DOI] [PubMed] [Google Scholar]
  17. Kamp D., Kardas E., Ritthaler W., Sandulache R., Schmucker R., Stern B. Comparative analysis of invertible DNA in phage genomes. Cold Spring Harb Symp Quant Biol. 1984;49:301–311. doi: 10.1101/sqb.1984.049.01.036. [DOI] [PubMed] [Google Scholar]
  18. Kanaar R., van de Putte P., Cozzarelli N. R. Gin-mediated DNA inversion: product structure and the mechanism of strand exchange. Proc Natl Acad Sci U S A. 1988 Feb;85(3):752–756. doi: 10.1073/pnas.85.3.752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kennedy K. E., Iida S., Meyer J., Stålhammar-Carlemalm M., Hiestand-Nauer R., Arber W. Genome fusion mediated by the site specific DNA inversion system of bacteriophage P1. Mol Gen Genet. 1983;189(3):413–421. doi: 10.1007/BF00325903. [DOI] [PubMed] [Google Scholar]
  20. Kitts P. A., Symington L. S., Dyson P., Sherratt D. J. Transposon-encoded site-specific recombination: nature of the Tn3 DNA sequences which constitute the recombination site res. EMBO J. 1983;2(7):1055–1060. doi: 10.1002/j.1460-2075.1983.tb01545.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Koch C., Kahmann R. Purification and properties of the Escherichia coli host factor required for inversion of the G segment in bacteriophage Mu. J Biol Chem. 1986 Nov 25;261(33):15673–15678. [PubMed] [Google Scholar]
  22. Krasnow M. A., Cozzarelli N. R. Site-specific relaxation and recombination by the Tn3 resolvase: recognition of the DNA path between oriented res sites. Cell. 1983 Apr;32(4):1313–1324. doi: 10.1016/0092-8674(83)90312-4. [DOI] [PubMed] [Google Scholar]
  23. Lange-Gustafson B. J., Nash H. A. Purification and properties of Int-h, a variant protein involved in site-specific recombination of bacteriophage lambda. J Biol Chem. 1984 Oct 25;259(20):12724–12732. [PubMed] [Google Scholar]
  24. Minton N. P. Improved plasmid vectors for the isolation of translational lac gene fusions. Gene. 1984 Nov;31(1-3):269–273. doi: 10.1016/0378-1119(84)90220-8. [DOI] [PubMed] [Google Scholar]
  25. Plasterk R. H., Ilmer T. A., Van de Putte P. Site-specific recombination by Gin of bacteriophage Mu: inversions and deletions. Virology. 1983 May;127(1):24–36. doi: 10.1016/0042-6822(83)90367-7. [DOI] [PubMed] [Google Scholar]
  26. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Scott T. N., Simon M. I. Genetic analysis of the mechanism of the Salmonella phase variation site specific recombination system. Mol Gen Genet. 1982;188(2):313–321. doi: 10.1007/BF00332694. [DOI] [PubMed] [Google Scholar]
  28. Silverman M., Zieg J., Mandel G., Simon M. Analysis of the functional components of the phase variation system. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):17–26. doi: 10.1101/sqb.1981.045.01.005. [DOI] [PubMed] [Google Scholar]
  29. van de Putte P., Plasterk R., Kuijpers A. A Mu gin complementing function and an invertible DNA region in Escherichia coli K-12 are situated on the genetic element e14. J Bacteriol. 1984 May;158(2):517–522. doi: 10.1128/jb.158.2.517-522.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]

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