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. 1997 Oct 1;25(19):3832–3839. doi: 10.1093/nar/25.19.3832

Stimulation of DNA inversion by FIS: evidence for enhancer-independent contacts with the Gin-gix complex.

A Deufel 1, T Hermann 1, R Kahmann 1, G Muskhelishvili 1
PMCID: PMC146962  PMID: 9380505

Abstract

Efficient DNA inversion catalysed by the invertase Gin requires the cis-acting recombinational enhancer and the Escherichia coliFIS protein. Binding of FIS bends the enhancer DNA and, on a negatively supercoiled DNA inversion substrate, facilitates the formation of a synaptic complex with specific topology. Previous studies have indicated that FIS-independent Gin mutants can be isolated which have lost the topological constraints imposed on the inversion reaction yet remain sensitive to the stimulatory effect of FIS. Whether the effect of FIS is purely architectural, or whether in addition direct protein contacts between Gin and FIS are required for efficient catalysis has remained an unresolved question. Here we show that FIS mutants impaired in DNA binding are capable of either positively or negatively affecting the inversion reaction both in vivo and in vitro. We further demonstrate that the mutant protein FIS K25E/V66A/M67T dramatically enhances the cleavage of recombination sites by FIS-independent Gin in an enhancer-independent manner. Our observations suggest that FIS plays a dual role in the inversion reaction and stimulates both the assembly of the synaptic complex as well as DNA strand cleavage.

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Selected References

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  1. Ball C. A., Johnson R. C. Multiple effects of Fis on integration and the control of lysogeny in phage lambda. J Bacteriol. 1991 Jul;173(13):4032–4038. doi: 10.1128/jb.173.13.4032-4038.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brosius J. Superpolylinkers in cloning and expression vectors. DNA. 1989 Dec;8(10):759–777. doi: 10.1089/dna.1989.8.759. [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. Bujard H., Gentz R., Lanzer M., Stueber D., Mueller M., Ibrahimi I., Haeuptle M. T., Dobberstein B. A T5 promoter-based transcription-translation system for the analysis of proteins in vitro and in vivo. Methods Enzymol. 1987;155:416–433. doi: 10.1016/0076-6879(87)55028-5. [DOI] [PubMed] [Google Scholar]
  5. Bétermier M., Poquet I., Alazard R., Chandler M. Involvement of Escherichia coli FIS protein in maintenance of bacteriophage mu lysogeny by the repressor: control of early transcription and inhibition of transposition. J Bacteriol. 1993 Jun;175(12):3798–3811. doi: 10.1128/jb.175.12.3798-3811.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bölker M., Kahmann R. The Escherichia coli regulatory protein OxyR discriminates between methylated and unmethylated states of the phage Mu mom promoter. EMBO J. 1989 Aug;8(8):2403–2410. doi: 10.1002/j.1460-2075.1989.tb08370.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cassler M. R., Grimwade J. E., Leonard A. C. Cell cycle-specific changes in nucleoprotein complexes at a chromosomal replication origin. EMBO J. 1995 Dec 1;14(23):5833–5841. doi: 10.1002/j.1460-2075.1995.tb00271.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Crisona N. J., Kanaar R., Gonzalez T. N., Zechiedrich E. L., Klippel A., Cozzarelli N. R. Processive recombination by wild-type gin and an enhancer-independent mutant. Insight into the mechanisms of recombination selectivity and strand exchange. J Mol Biol. 1994 Oct 28;243(3):437–457. doi: 10.1006/jmbi.1994.1671. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Gille H., Egan J. B., Roth A., Messer W. The FIS protein binds and bends the origin of chromosomal DNA replication, oriC, of Escherichia coli. Nucleic Acids Res. 1991 Aug 11;19(15):4167–4172. doi: 10.1093/nar/19.15.4167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. González-Gil G., Bringmann P., Kahmann R. FIS is a regulator of metabolism in Escherichia coli. Mol Microbiol. 1996 Oct;22(1):21–29. doi: 10.1111/j.1365-2958.1996.tb02652.x. [DOI] [PubMed] [Google Scholar]
  12. Hamilton C. M., Aldea M., Washburn B. K., Babitzke P., Kushner S. R. New method for generating deletions and gene replacements in Escherichia coli. J Bacteriol. 1989 Sep;171(9):4617–4622. doi: 10.1128/jb.171.9.4617-4622.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Haykinson M. J., Johnson L. M., Soong J., Johnson R. C. The Hin dimer interface is critical for Fis-mediated activation of the catalytic steps of site-specific DNA inversion. Curr Biol. 1996 Feb 1;6(2):163–177. doi: 10.1016/s0960-9822(02)00449-9. [DOI] [PubMed] [Google Scholar]
  14. Heichman K. A., Johnson R. C. The Hin invertasome: protein-mediated joining of distant recombination sites at the enhancer. Science. 1990 Aug 3;249(4968):511–517. doi: 10.1126/science.2166334. [DOI] [PubMed] [Google Scholar]
  15. Heichman K. A., Moskowitz I. P., Johnson R. C. Configuration of DNA strands and mechanism of strand exchange in the Hin invertasome as revealed by analysis of recombinant knots. Genes Dev. 1991 Sep;5(9):1622–1634. doi: 10.1101/gad.5.9.1622. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Hughes R. E., Hatfull G. F., Rice P., Steitz T. A., Grindley N. D. Cooperativity mutants of the gamma delta resolvase identify an essential interdimer interaction. Cell. 1990 Dec 21;63(6):1331–1338. doi: 10.1016/0092-8674(90)90428-h. [DOI] [PubMed] [Google Scholar]
  18. Hübner P., Haffter P., Iida S., Arber W. Bent DNA is needed for recombinational enhancer activity in the site-specific recombination system Cin of bacteriophage P1. The role of FIS protein. J Mol Biol. 1989 Feb 5;205(3):493–500. doi: 10.1016/0022-2836(89)90220-9. [DOI] [PubMed] [Google Scholar]
  19. Johnson R. C., Ball C. A., Pfeffer D., Simon M. I. Isolation of the gene encoding the Hin recombinational enhancer binding protein. Proc Natl Acad Sci U S A. 1988 May;85(10):3484–3488. doi: 10.1073/pnas.85.10.3484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Johnson R. C., Bruist M. F. Intermediates in Hin-mediated DNA inversion: a role for Fis and the recombinational enhancer in the strand exchange reaction. EMBO J. 1989 May;8(5):1581–1590. doi: 10.1002/j.1460-2075.1989.tb03542.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Kanaar R., Klippel A., Shekhtman E., Dungan J. M., Kahmann R., Cozzarelli N. R. Processive recombination by the phage Mu Gin system: implications for the mechanisms of DNA strand exchange, DNA site alignment, and enhancer action. Cell. 1990 Jul 27;62(2):353–366. doi: 10.1016/0092-8674(90)90372-l. [DOI] [PubMed] [Google Scholar]
  24. Kanaar R., van de Putte P., Cozzarelli N. R. Gin-mediated recombination of catenated and knotted DNA substrates: implications for the mechanism of interaction between cis-acting sites. Cell. 1989 Jul 14;58(1):147–159. doi: 10.1016/0092-8674(89)90411-x. [DOI] [PubMed] [Google Scholar]
  25. Klippel A., Cloppenborg K., Kahmann R. Isolation and characterization of unusual gin mutants. EMBO J. 1988 Dec 1;7(12):3983–3989. doi: 10.1002/j.1460-2075.1988.tb03286.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Klippel A., Kanaar R., Kahmann R., Cozzarelli N. R. Analysis of strand exchange and DNA binding of enhancer-independent Gin recombinase mutants. EMBO J. 1993 Mar;12(3):1047–1057. doi: 10.1002/j.1460-2075.1993.tb05746.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Klippel A., Mertens G., Patschinsky T., Kahmann R. The DNA invertase Gin of phage Mu: formation of a covalent complex with DNA via a phosphoserine at amino acid position 9. EMBO J. 1988 Apr;7(4):1229–1237. doi: 10.1002/j.1460-2075.1988.tb02935.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Koch C., Ninnemann O., Fuss H., Kahmann R. The N-terminal part of the E.coli DNA binding protein FIS is essential for stimulating site-specific DNA inversion but is not required for specific DNA binding. Nucleic Acids Res. 1991 Nov 11;19(21):5915–5922. doi: 10.1093/nar/19.21.5915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Koch C., Vandekerckhove J., Kahmann R. Escherichia coli host factor for site-specific DNA inversion: cloning and characterization of the fis gene. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4237–4241. doi: 10.1073/pnas.85.12.4237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kostrewa D., Granzin J., Koch C., Choe H. W., Raghunathan S., Wolf W., Labahn J., Kahmann R., Saenger W. Three-dimensional structure of the E. coli DNA-binding protein FIS. Nature. 1991 Jan 10;349(6305):178–180. doi: 10.1038/349178a0. [DOI] [PubMed] [Google Scholar]
  32. Kostrewa D., Granzin J., Stock D., Choe H. W., Labahn J., Saenger W. Crystal structure of the factor for inversion stimulation FIS at 2.0 A resolution. J Mol Biol. 1992 Jul 5;226(1):209–226. doi: 10.1016/0022-2836(92)90134-6. [DOI] [PubMed] [Google Scholar]
  33. Kuipers O. P., Boot H. J., de Vos W. M. Improved site-directed mutagenesis method using PCR. Nucleic Acids Res. 1991 Aug 25;19(16):4558–4558. doi: 10.1093/nar/19.16.4558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Landt O., Grunert H. P., Hahn U. A general method for rapid site-directed mutagenesis using the polymerase chain reaction. Gene. 1990 Nov 30;96(1):125–128. doi: 10.1016/0378-1119(90)90351-q. [DOI] [PubMed] [Google Scholar]
  35. Lanzer M., Bujard H. Promoters largely determine the efficiency of repressor action. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8973–8977. doi: 10.1073/pnas.85.23.8973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lazarus L. R., Travers A. A. The Escherichia coli FIS protein is not required for the activation of tyrT transcription on entry into exponential growth. EMBO J. 1993 Jun;12(6):2483–2494. doi: 10.1002/j.1460-2075.1993.tb05903.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Merker P., Muskhelishvili G., Deufel A., Rusch K., Kahmann R. Role of Gin and FIS in site-specific recombination. Cold Spring Harb Symp Quant Biol. 1993;58:505–513. doi: 10.1101/sqb.1993.058.01.057. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Mertens G., Klippel A., Fuss H., Blöcker H., Frank R., Kahmann R. Site-specific recombination in bacteriophage Mu: characterization of binding sites for the DNA invertase Gin. EMBO J. 1988 Apr;7(4):1219–1227. doi: 10.1002/j.1460-2075.1988.tb02934.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Muskhelishvili G., Travers A. A., Heumann H., Kahmann R. FIS and RNA polymerase holoenzyme form a specific nucleoprotein complex at a stable RNA promoter. EMBO J. 1995 Apr 3;14(7):1446–1452. doi: 10.1002/j.1460-2075.1995.tb07131.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Nilsson L., Vanet A., Vijgenboom E., Bosch L. The role of FIS in trans activation of stable RNA operons of E. coli. EMBO J. 1990 Mar;9(3):727–734. doi: 10.1002/j.1460-2075.1990.tb08166.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. North A. K., Kustu S. Mutant forms of the enhancer-binding protein NtrC can activate transcription from solution. J Mol Biol. 1997 Mar 21;267(1):17–36. doi: 10.1006/jmbi.1996.0838. [DOI] [PubMed] [Google Scholar]
  43. Oka A., Sugisaki H., Takanami M. Nucleotide sequence of the kanamycin resistance transposon Tn903. J Mol Biol. 1981 Apr 5;147(2):217–226. doi: 10.1016/0022-2836(81)90438-1. [DOI] [PubMed] [Google Scholar]
  44. Osuna R., Finkel S. E., Johnson R. C. Identification of two functional regions in Fis: the N-terminus is required to promote Hin-mediated DNA inversion but not lambda excision. EMBO J. 1991 Jun;10(6):1593–1603. doi: 10.1002/j.1460-2075.1991.tb07680.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Pan C. Q., Feng J. A., Finkel S. E., Landgraf R., Sigman D., Johnson R. C. Structure of the Escherichia coli Fis-DNA complex probed by protein conjugated with 1,10-phenanthroline copper(I) complex. Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1721–1725. doi: 10.1073/pnas.91.5.1721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Pan C. Q., Finkel S. E., Cramton S. E., Feng J. A., Sigman D. S., Johnson R. C. Variable structures of Fis-DNA complexes determined by flanking DNA-protein contacts. J Mol Biol. 1996 Dec 13;264(4):675–695. doi: 10.1006/jmbi.1996.0669. [DOI] [PubMed] [Google Scholar]
  47. Reed R. R., Grindley N. D. Transposon-mediated site-specific recombination in vitro: DNA cleavage and protein-DNA linkage at the recombination site. Cell. 1981 Sep;25(3):721–728. doi: 10.1016/0092-8674(81)90179-3. [DOI] [PubMed] [Google Scholar]
  48. Ross W., Thompson J. F., Newlands J. T., Gourse R. L. E.coli Fis protein activates ribosomal RNA transcription in vitro and in vivo. EMBO J. 1990 Nov;9(11):3733–3742. doi: 10.1002/j.1460-2075.1990.tb07586.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sanderson M. R., Freemont P. S., Rice P. A., Goldman A., Hatfull G. F., Grindley N. D., Steitz T. A. The crystal structure of the catalytic domain of the site-specific recombination enzyme gamma delta resolvase at 2.7 A resolution. Cell. 1990 Dec 21;63(6):1323–1329. doi: 10.1016/0092-8674(90)90427-g. [DOI] [PubMed] [Google Scholar]
  50. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  51. Spee J. H., de Vos W. M., Kuipers O. P. Efficient random mutagenesis method with adjustable mutation frequency by use of PCR and dITP. Nucleic Acids Res. 1993 Feb 11;21(3):777–778. doi: 10.1093/nar/21.3.777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Thompson J. F., Landy A. Empirical estimation of protein-induced DNA bending angles: applications to lambda site-specific recombination complexes. Nucleic Acids Res. 1988 Oct 25;16(20):9687–9705. doi: 10.1093/nar/16.20.9687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Thompson J. F., Moitoso de Vargas L., Koch C., Kahmann R., Landy A. Cellular factors couple recombination with growth phase: characterization of a new component in the lambda site-specific recombination pathway. Cell. 1987 Sep 11;50(6):901–908. doi: 10.1016/0092-8674(87)90516-2. [DOI] [PubMed] [Google Scholar]
  55. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  56. Weinreich M. D., Reznikoff W. S. Fis plays a role in Tn5 and IS50 transposition. J Bacteriol. 1992 Jul;174(14):4530–4537. doi: 10.1128/jb.174.14.4530-4537.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Yuan H. S., Finkel S. E., Feng J. A., Kaczor-Grzeskowiak M., Johnson R. C., Dickerson R. E. The molecular structure of wild-type and a mutant Fis protein: relationship between mutational changes and recombinational enhancer function or DNA binding. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9558–9562. doi: 10.1073/pnas.88.21.9558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. van Drunen C. M., van Zuylen C., Mientjes E. J., Goosen N., van de Putte P. Inhibition of bacteriophage Mu transposition by Mu repressor and Fis. Mol Microbiol. 1993 Oct;10(2):293–298. doi: 10.1111/j.1365-2958.1993.tb01955.x. [DOI] [PubMed] [Google Scholar]
  59. van de Putte P., Goosen N. DNA inversions in phages and bacteria. Trends Genet. 1992 Dec;8(12):457–462. doi: 10.1016/0168-9525(92)90331-w. [DOI] [PubMed] [Google Scholar]

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