Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1988 Apr;7(4):1219–1227. doi: 10.1002/j.1460-2075.1988.tb02934.x

Site-specific recombination in bacteriophage Mu: characterization of binding sites for the DNA invertase Gin.

G Mertens 1, A Klippel 1, H Fuss 1, H Blöcker 1, R Frank 1, R Kahmann 1
PMCID: PMC454459  PMID: 3042381

Abstract

Site-specific DNA inversion in phage Mu is catalysed by the phage-encoded DNA invertase Gin and a host factor FIS. We demonstrate that purified Gin protein binds specifically to 34-bp sequences that flank the G segment as inverted repeats. Each inverted repeat (IR) contains two binding sites for Gin which have to be arranged in a specific configuration to constitute a recombinogenic site. While one of these sites is bound when present alone, the other site is bound only in conjunction with the first one, suggesting cooperative binding. In addition to the sites within the IR, Gin binds with lower affinity to AT-rich sequences adjacent to the IR. We demonstrate that these sites do not participate in the inversion reaction. The IR itself can be shortened to 25 bp without effect on inversion frequency. Using gel mobility shift experiments on circular permuted fragments containing the IR we show that Gin bends DNA upon binding. We discuss the possibility that DNA bending is related to the formation of a productive synaptic complex.

Full text

PDF
1219

Images in this article

1220Image
on p.1220
1221Image
on p.1221
1223Image
on p.1223

Selected References

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

  1. Abdel-Meguid S. S., Grindley N. D., Templeton N. S., Steitz T. A. Cleavage of the site-specific recombination protein gamma delta resolvase: the smaller of two fragments binds DNA specifically. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2001–2005. doi: 10.1073/pnas.81.7.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beck E., Ludwig G., Auerswald E. A., Reiss B., Schaller H. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene. 1982 Oct;19(3):327–336. doi: 10.1016/0378-1119(82)90023-3. [DOI] [PubMed] [Google Scholar]
  3. Frank R., Heikens W., Heisterberg-Moutsis G., Blöcker H. A new general approach for the simultaneous chemical synthesis of large numbers of oligonucleotides: segmental solid supports. Nucleic Acids Res. 1983 Jul 11;11(13):4365–4377. doi: 10.1093/nar/11.13.4365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Grundy F. J., Howe M. M. Involvement of the invertible G segment in bacteriophage mu tail fiber biosynthesis. Virology. 1984 Apr 30;134(2):296–317. doi: 10.1016/0042-6822(84)90299-x. [DOI] [PubMed] [Google Scholar]
  6. Hatfull G. F., Noble S. M., Grindley N. D. The gamma delta resolvase induces an unusual DNA structure at the recombinational crossover point. Cell. 1987 Apr 10;49(1):103–110. doi: 10.1016/0092-8674(87)90760-4. [DOI] [PubMed] [Google Scholar]
  7. Hertzberg R. P., Dervan P. B. Cleavage of DNA with methidiumpropyl-EDTA-iron(II): reaction conditions and product analyses. Biochemistry. 1984 Aug 14;23(17):3934–3945. doi: 10.1021/bi00312a022. [DOI] [PubMed] [Google Scholar]
  8. Hoess R. H., Wierzbicki A., Abremski K. The role of the loxP spacer region in P1 site-specific recombination. Nucleic Acids Res. 1986 Mar 11;14(5):2287–2300. doi: 10.1093/nar/14.5.2287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Iida S., Hiestand-Nauer R. Localized conversion at the crossover sequences in the site-specific DNA inversion system of bacteriophage P1. Cell. 1986 Apr 11;45(1):71–79. doi: 10.1016/0092-8674(86)90539-8. [DOI] [PubMed] [Google Scholar]
  11. Iida S., Hiestand-Nauer R. Role of the central dinucleotide at the crossover sites for the selection of quasi sites in DNA inversion mediated by the site-specific Cin recombinase of phage P1. Mol Gen Genet. 1987 Jul;208(3):464–468. doi: 10.1007/BF00328140. [DOI] [PubMed] [Google Scholar]
  12. Johnson R. C., Bruist M. B., Glaccum M. B., Simon M. I. In vitro analysis of Hin-mediated site-specific recombination. Cold Spring Harb Symp Quant Biol. 1984;49:751–760. doi: 10.1101/sqb.1984.049.01.085. [DOI] [PubMed] [Google Scholar]
  13. Johnson R. C., Bruist M. F., Simon M. I. Host protein requirements for in vitro site-specific DNA inversion. Cell. 1986 Aug 15;46(4):531–539. doi: 10.1016/0092-8674(86)90878-0. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. 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]
  17. Kahmann R., Rudt F., Mertens G. Substrate and enzyme requirements for in vitro site-specific recombination in bacteriophage mu. Cold Spring Harb Symp Quant Biol. 1984;49:285–294. doi: 10.1101/sqb.1984.049.01.034. [DOI] [PubMed] [Google Scholar]
  18. Kamp D., Kahmann R., Zipser D., Broker T. R., Chow L. T. Inversion of the G DNA segment of phage Mu controls phage infectivity. Nature. 1978 Feb 9;271(5645):577–580. doi: 10.1038/271577a0. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Mertens G., Fuss H., Kahmann R. Purification and properties of the DNA invertase gin encoded by bacteriophage Mu. J Biol Chem. 1986 Nov 25;261(33):15668–15672. [PubMed] [Google Scholar]
  22. Mertens G., Hoffmann A., Blöcker H., Frank R., Kahmann R. Gin-mediated site-specific recombination in bacteriophage Mu DNA: overproduction of the protein and inversion in vitro. EMBO J. 1984 Oct;3(10):2415–2421. doi: 10.1002/j.1460-2075.1984.tb02148.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Plasterk R. H., Brinkman A., van de Putte P. DNA inversions in the chromosome of Escherichia coli and in bacteriophage Mu: relationship to other site-specific recombination systems. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5355–5358. doi: 10.1073/pnas.80.17.5355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Plasterk R. H., Kanaar R., van de Putte P. A genetic switch in vitro: DNA inversion by Gin protein of phage Mu. Proc Natl Acad Sci U S A. 1984 May;81(9):2689–2692. doi: 10.1073/pnas.81.9.2689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Plasterk R. H., Van de Putte P. Genetic switches by DNA inversions in prokaryotes. Biochim Biophys Acta. 1984 Jun 16;782(2):111–119. doi: 10.1016/0167-4781(84)90013-7. [DOI] [PubMed] [Google Scholar]
  26. Sadowski P. Site-specific recombinases: changing partners and doing the twist. J Bacteriol. 1986 Feb;165(2):341–347. doi: 10.1128/jb.165.2.341-347.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Schmucker R., Ritthaler W., Stern B., Kamp D. DNA inversion in bacteriophage Mu: characterization of the inversion site. J Gen Virol. 1986 Jun;67(Pt 6):1123–1133. doi: 10.1099/0022-1317-67-6-1123. [DOI] [PubMed] [Google Scholar]
  29. Wu H. M., Crothers D. M. The locus of sequence-directed and protein-induced DNA bending. Nature. 1984 Apr 5;308(5959):509–513. doi: 10.1038/308509a0. [DOI] [PubMed] [Google Scholar]
  30. van de Putte P., Cramer S., Giphart-Gassler M. Invertible DNA determines host specificity of bacteriophage mu. Nature. 1980 Jul 17;286(5770):218–222. doi: 10.1038/286218a0. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES