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. 1994 Apr 15;13(8):1844–1855. doi: 10.1002/j.1460-2075.1994.tb06453.x

Xer-mediated site-specific recombination at cer generates Holliday junctions in vivo.

R McCulloch 1, L W Coggins 1, S D Colloms 1, D J Sherratt 1
PMCID: PMC395024  PMID: 8168483

Abstract

Normal segregation of the Escherichia coli chromosome and stable inheritance of multicopy plasmids such as ColE1 requires the Xer site-specific recombination system. Two putative lambda integrase family recombinases, XerC and XerD, participate in the recombination reactions. We have constructed an E. coli strain in which the expression of xerC can be tightly regulated, thereby allowing the analysis of controlled recombination reactions in vivo. Xer-mediated recombination in this strain generates Holliday junction-containing DNA molecules in which a specific pair of strands has been exchanged in addition to complete recombinant products. This suggests that Xer site-specific recombination utilizes a strand exchange mechanism similar or identical to that of other members of the lambda integrase family of recombination systems. The controlled in vivo recombination reaction at cer requires recombinase and two accessory proteins, ArgR and PepA. Generation of Holliday junctions and recombinant products is equally efficient in RuvC- and RuvC+ cells, and in cells containing a multicopy RuvC+ plasmid. Controlled XerC expression is also used to analyse the efficiency of recombination between variant cer sites containing sequence alterations and heterologies within their central regions.

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Selected References

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

  1. Adams D. E., Bliska J. B., Cozzarelli N. R. Cre-lox recombination in Escherichia coli cells. Mechanistic differences from the in vitro reaction. J Mol Biol. 1992 Aug 5;226(3):661–673. doi: 10.1016/0022-2836(92)90623-r. [DOI] [PubMed] [Google Scholar]
  2. Andrews B. J., Beatty L. G., Sadowski P. D. Isolation of intermediates in the binding of the FLP recombinase of the yeast plasmid 2-micron circle to its target sequence. J Mol Biol. 1987 Jan 20;193(2):345–358. doi: 10.1016/0022-2836(87)90223-3. [DOI] [PubMed] [Google Scholar]
  3. Bachmann B. J. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol Rev. 1972 Dec;36(4):525–557. doi: 10.1128/br.36.4.525-557.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bauer C. E., Gardner J. F., Gumport R. I., Weisberg R. A. The effect of attachment site mutations on strand exchange in bacteriophage lambda site-specific recombination. Genetics. 1989 Aug;122(4):727–736. doi: 10.1093/genetics/122.4.727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blakely G., Colloms S., May G., Burke M., Sherratt D. Escherichia coli XerC recombinase is required for chromosomal segregation at cell division. New Biol. 1991 Aug;3(8):789–798. [PubMed] [Google Scholar]
  6. Blakely G., May G., McCulloch R., Arciszewska L. K., Burke M., Lovett S. T., Sherratt D. J. Two related recombinases are required for site-specific recombination at dif and cer in E. coli K12. Cell. 1993 Oct 22;75(2):351–361. doi: 10.1016/0092-8674(93)80076-q. [DOI] [PubMed] [Google Scholar]
  7. Bliska J. B., Benjamin H. W., Cozzarelli N. R. Mechanism of Tn3 resolvase recombination in vivo. J Biol Chem. 1991 Feb 5;266(4):2041–2047. [PubMed] [Google Scholar]
  8. Bliska J. B., Cozzarelli N. R. Use of site-specific recombination as a probe of DNA structure and metabolism in vivo. J Mol Biol. 1987 Mar 20;194(2):205–218. doi: 10.1016/0022-2836(87)90369-x. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Colloms S. D., Sykora P., Szatmari G., Sherratt D. J. Recombination at ColE1 cer requires the Escherichia coli xerC gene product, a member of the lambda integrase family of site-specific recombinases. J Bacteriol. 1990 Dec;172(12):6973–6980. doi: 10.1128/jb.172.12.6973-6980.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Connolly B., Parsons C. A., Benson F. E., Dunderdale H. J., Sharples G. J., Lloyd R. G., West S. C. Resolution of Holliday junctions in vitro requires the Escherichia coli ruvC gene product. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6063–6067. doi: 10.1073/pnas.88.14.6063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Craig N. L., Nash H. A. The mechanism of phage lambda site-specific recombination: site-specific breakage of DNA by Int topoisomerase. Cell. 1983 Dec;35(3 Pt 2):795–803. doi: 10.1016/0092-8674(83)90112-5. [DOI] [PubMed] [Google Scholar]
  13. Dunderdale H. J., Benson F. E., Parsons C. A., Sharples G. J., Lloyd R. G., West S. C. Formation and resolution of recombination intermediates by E. coli RecA and RuvC proteins. Nature. 1991 Dec 19;354(6354):506–510. doi: 10.1038/354506a0. [DOI] [PubMed] [Google Scholar]
  14. Echols H., Green L. Some properties of site-specific and general recombination inferred from int-initiated exchanges by bacteriophage lambda. Genetics. 1979 Oct;93(2):297–307. doi: 10.1093/genetics/93.2.297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Enquist L. W., Nash H., Weisberg R. A. Strand exchange in site-specific recombination. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1363–1367. doi: 10.1073/pnas.76.3.1363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hakkaart M. J., van den Elzen P. J., Veltkamp E., Nijkamp H. J. Maintenance of multicopy plasmid Clo DF13 in E. coli cells: evidence for site-specific recombination at parB. Cell. 1984 Jan;36(1):203–209. doi: 10.1016/0092-8674(84)90090-4. [DOI] [PubMed] [Google Scholar]
  17. Heery D. M., Gannon F., Powell R. A simple method for subcloning DNA fragments from gel slices. Trends Genet. 1990 Jun;6(6):173–173. doi: 10.1016/0168-9525(90)90158-3. [DOI] [PubMed] [Google Scholar]
  18. Hoess R. H., Abremski K. Mechanism of strand cleavage and exchange in the Cre-lox site-specific recombination system. J Mol Biol. 1985 Feb 5;181(3):351–362. doi: 10.1016/0022-2836(85)90224-4. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Hoess R., Wierzbicki A., Abremski K. Isolation and characterization of intermediates in site-specific recombination. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6840–6844. doi: 10.1073/pnas.84.19.6840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  22. Iwasaki H., Takahagi M., Shiba T., Nakata A., Shinagawa H. Escherichia coli RuvC protein is an endonuclease that resolves the Holliday structure. EMBO J. 1991 Dec;10(13):4381–4389. doi: 10.1002/j.1460-2075.1991.tb05016.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jayaram M., Crain K. L., Parsons R. L., Harshey R. M. Holliday junctions in FLP recombination: resolution by step-arrest mutants of FLP protein. Proc Natl Acad Sci U S A. 1988 Nov;85(21):7902–7906. doi: 10.1073/pnas.85.21.7902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kitts P. A., Nash H. A. An intermediate in the phage lambda site-specific recombination reaction is revealed by phosphorothioate substitution in DNA. Nucleic Acids Res. 1988 Jul 25;16(14B):6839–6856. doi: 10.1093/nar/16.14.6839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kitts P. A., Nash H. A. Bacteriophage lambda site-specific recombination proceeds with a defined order of strand exchanges. J Mol Biol. 1988 Nov 5;204(1):95–107. doi: 10.1016/0022-2836(88)90602-x. [DOI] [PubMed] [Google Scholar]
  26. Kitts P. A., Nash H. A. Homology-dependent interactions in phage lambda site-specific recombination. Nature. 1987 Sep 24;329(6137):346–348. doi: 10.1038/329346a0. [DOI] [PubMed] [Google Scholar]
  27. Kuempel P. L., Henson J. M., Dircks L., Tecklenburg M., Lim D. F. dif, a recA-independent recombination site in the terminus region of the chromosome of Escherichia coli. New Biol. 1991 Aug;3(8):799–811. [PubMed] [Google Scholar]
  28. Landy A. Dynamic, structural, and regulatory aspects of lambda site-specific recombination. Annu Rev Biochem. 1989;58:913–949. doi: 10.1146/annurev.bi.58.070189.004405. [DOI] [PubMed] [Google Scholar]
  29. Lovett S. T., Kolodner R. D. Nucleotide sequence of the Escherichia coli recJ chromosomal region and construction of recJ-overexpression plasmids. J Bacteriol. 1991 Jan;173(1):353–364. doi: 10.1128/jb.173.1.353-364.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. MAAS W. K. Studies on repression of arginine biosynthesis in Escherichia coli. Cold Spring Harb Symp Quant Biol. 1961;26:183–191. doi: 10.1101/sqb.1961.026.01.023. [DOI] [PubMed] [Google Scholar]
  31. McLeod M., Craft S., Broach J. R. Identification of the crossover site during FLP-mediated recombination in the Saccharomyces cerevisiae plasmid 2 microns circle. Mol Cell Biol. 1986 Oct;6(10):3357–3367. doi: 10.1128/mcb.6.10.3357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Meyer-Leon L., Huang L. C., Umlauf S. W., Cox M. M., Inman R. B. Holliday intermediates and reaction by-products in FLP protein-promoted site-specific recombination. Mol Cell Biol. 1988 Sep;8(9):3784–3796. doi: 10.1128/mcb.8.9.3784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Meyer-Leon L., Inman R. B., Cox M. M. Characterization of Holliday structures in FLP protein-promoted site-specific recombination. Mol Cell Biol. 1990 Jan;10(1):235–242. doi: 10.1128/mcb.10.1.235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nunes-Düby S. E., Matsumoto L., Landy A. Site-specific recombination intermediates trapped with suicide substrates. Cell. 1987 Aug 28;50(5):779–788. doi: 10.1016/0092-8674(87)90336-9. [DOI] [PubMed] [Google Scholar]
  35. Richaud C., Higgins W., Mengin-Lecreulx D., Stragier P. Molecular cloning, characterization, and chromosomal localization of dapF, the Escherichia coli gene for diaminopimelate epimerase. J Bacteriol. 1987 Apr;169(4):1454–1459. doi: 10.1128/jb.169.4.1454-1459.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Richaud C., Printz C. Nucleotide sequence of the dapF gene and flanking regions from Escherichia coli K12. Nucleic Acids Res. 1988 Nov 11;16(21):10367–10367. doi: 10.1093/nar/16.21.10367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Senecoff J. F., Cox M. M. Directionality in FLP protein-promoted site-specific recombination is mediated by DNA-DNA pairing. J Biol Chem. 1986 Jun 5;261(16):7380–7386. [PubMed] [Google Scholar]
  38. Sharples G. J., Lloyd R. G. Resolution of Holliday junctions in Escherichia coli: identification of the ruvC gene product as a 19-kilodalton protein. J Bacteriol. 1991 Dec;173(23):7711–7715. doi: 10.1128/jb.173.23.7711-7715.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stark W. M., Boocock M. R., Sherratt D. J. Catalysis by site-specific recombinases. Trends Genet. 1992 Dec;8(12):432–439. [PubMed] [Google Scholar]
  40. Stirling C. J., Colloms S. D., Collins J. F., Szatmari G., Sherratt D. J. xerB, an Escherichia coli gene required for plasmid ColE1 site-specific recombination, is identical to pepA, encoding aminopeptidase A, a protein with substantial similarity to bovine lens leucine aminopeptidase. EMBO J. 1989 May;8(5):1623–1627. doi: 10.1002/j.1460-2075.1989.tb03547.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Stirling C. J., Stewart G., Sherratt D. J. Multicopy plasmid stability in Escherichia coli requires host-encoded functions that lead to plasmid site-specific recombination. Mol Gen Genet. 1988 Sep;214(1):80–84. doi: 10.1007/BF00340183. [DOI] [PubMed] [Google Scholar]
  42. Stirling C. J., Szatmari G., Stewart G., Smith M. C., Sherratt D. J. The arginine repressor is essential for plasmid-stabilizing site-specific recombination at the ColE1 cer locus. EMBO J. 1988 Dec 20;7(13):4389–4395. doi: 10.1002/j.1460-2075.1988.tb03338.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Summers D. K. Derivatives of ColE1 cer show altered topological specificity in site-specific recombination. EMBO J. 1989 Jan;8(1):309–315. doi: 10.1002/j.1460-2075.1989.tb03378.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Summers D. K., Sherratt D. J. Multimerization of high copy number plasmids causes instability: CoIE1 encodes a determinant essential for plasmid monomerization and stability. Cell. 1984 Apr;36(4):1097–1103. doi: 10.1016/0092-8674(84)90060-6. [DOI] [PubMed] [Google Scholar]
  45. Summers D. K., Sherratt D. J. Resolution of ColE1 dimers requires a DNA sequence implicated in the three-dimensional organization of the cer site. EMBO J. 1988 Mar;7(3):851–858. doi: 10.1002/j.1460-2075.1988.tb02884.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Summers D., Yaish S., Archer J., Sherratt D. Multimer resolution systems of ColE1 and ColK: localisation of the crossover site. Mol Gen Genet. 1985;201(2):334–338. doi: 10.1007/BF00425680. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Takahagi M., Iwasaki H., Nakata A., Shinagawa H. Molecular analysis of the Escherichia coli ruvC gene, which encodes a Holliday junction-specific endonuclease. J Bacteriol. 1991 Sep;173(18):5747–5753. doi: 10.1128/jb.173.18.5747-5753.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Valenzuela M. S., Inman R. B. Visualization of a novel junction in bacteriophage lambda DNA. Proc Natl Acad Sci U S A. 1975 Aug;72(8):3024–3028. doi: 10.1073/pnas.72.8.3024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Weisberg R. A., Enquist L. W., Foeller C., Landy A. Role for DNA homology in site-specific recombination. The isolation and characterization of a site affinity mutant of coliphage lambda. J Mol Biol. 1983 Oct 25;170(2):319–342. doi: 10.1016/s0022-2836(83)80151-x. [DOI] [PubMed] [Google Scholar]
  51. Winans S. C., Elledge S. J., Krueger J. H., Walker G. C. Site-directed insertion and deletion mutagenesis with cloned fragments in Escherichia coli. J Bacteriol. 1985 Mar;161(3):1219–1221. doi: 10.1128/jb.161.3.1219-1221.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. de Massy B., Dorgai L., Weisberg R. A. Mutations of the phage lambda attachment site alter the directionality of resolution of Holliday structures. EMBO J. 1989 May;8(5):1591–1599. doi: 10.1002/j.1460-2075.1989.tb03543.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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