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. 1992 Dec 11;20(23):6159–6166. doi: 10.1093/nar/20.23.6159

Recombination of nicked DNA knots by gamma delta resolvase suggests a variant model for the mechanism of strand exchange.

P Dröge 1
PMCID: PMC334499  PMID: 1335567

Abstract

Fast and efficient recombination catalyzed by gamma delta resolvase in vitro requires negative DNA supercoiling of plasmid substrates. The current model for recombination suggests that supercoiling is required to drive DNA strand exchange within a synaptic complex by 'simple rotation' of DNA-linked resolvase promoters. Surprisingly, DNA knots are recombined efficiently in the absence of supercoiling, whereby the rate of recombination increases with the number of irreducible DNA segment crossings, or nodes, within each substrate knot. Recombination products contain three knot nodes less than substrates, suggesting that a reduction in writhe drives the reaction. However, the proposed protomer rotation model predicts that writhe is not altered during the process of strand transfer but, instead, is reduced only when a synaptic complex disassembles after strand exchange. I present evidence that recombination of knotted and of linear substrates coincides with a disassembly of synaptic complexes. The results lead to a variant model for strand exchange on non-supercoiled substrates in which a specific disassembly of the synaptic complex, triggered by a reduction in writhe, guides the cleaved DNA into the recombinant configuration.

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Selected References

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  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. Bednarz A. L., Boocock M. R., Sherratt D. J. Determinants of correct res site alignment in site-specific recombination by Tn3 resolvase. Genes Dev. 1990 Dec;4(12B):2366–2375. doi: 10.1101/gad.4.12b.2366. [DOI] [PubMed] [Google Scholar]
  3. Benjamin H. W., Cozzarelli N. R. Geometric arrangements of Tn3 resolvase sites. J Biol Chem. 1990 Apr 15;265(11):6441–6447. [PubMed] [Google Scholar]
  4. 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]
  5. Benjamin H. W., Matzuk M. M., Krasnow M. A., Cozzarelli N. R. Recombination site selection by Tn3 resolvase: topological tests of a tracking mechanism. Cell. 1985 Jan;40(1):147–158. doi: 10.1016/0092-8674(85)90318-6. [DOI] [PubMed] [Google Scholar]
  6. Castell S. E., Halford S. E. DNA supercoiling determines the activation energy barrier for site specific recombination by Tn21 resolvase. Nucleic Acids Res. 1989 Sep 12;17(17):7045–7058. doi: 10.1093/nar/17.17.7045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Craigie R., Mizuuchi K. Role of DNA topology in Mu transposition: mechanism of sensing the relative orientation of two DNA segments. Cell. 1986 Jun 20;45(6):793–800. doi: 10.1016/0092-8674(86)90554-4. [DOI] [PubMed] [Google Scholar]
  8. Dröge P., Cozzarelli N. R. Recombination of knotted substrates by Tn3 resolvase. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6062–6066. doi: 10.1073/pnas.86.16.6062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dröge P., Cozzarelli N. R. Topological structure of DNA knots and catenanes. Methods Enzymol. 1992;212:120–130. doi: 10.1016/0076-6879(92)12008-e. [DOI] [PubMed] [Google Scholar]
  10. Dröge P., Hatfull G. F., Grindley N. D., Cozzarelli N. R. The two functional domains of gamma delta resolvase act on the same recombination site: implications for the mechanism of strand exchange. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5336–5340. doi: 10.1073/pnas.87.14.5336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grindley N. D., Lauth M. R., Wells R. G., Wityk R. J., Salvo J. J., Reed R. R. Transposon-mediated site-specific recombination: identification of three binding sites for resolvase at the res sites of gamma delta and Tn3. Cell. 1982 Aug;30(1):19–27. doi: 10.1016/0092-8674(82)90007-1. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Hatfull G. F., Grindley N. D. Analysis of gamma delta resolvase mutants in vitro: evidence for an interaction between serine-10 of resolvase and site I of res. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5429–5433. doi: 10.1073/pnas.83.15.5429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hatfull G. F., Sanderson M. R., Freemont P. S., Raccuia P. R., Grindley N. D., Steitz T. A. Preparation of heavy-atom derivatives using site-directed mutagenesis. Introduction of cysteine residues into gamma delta resolvase. J Mol Biol. 1989 Aug 20;208(4):661–667. doi: 10.1016/0022-2836(89)90156-3. [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. 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]
  17. 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]
  18. 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]
  19. Matsuoka M., Nagawa F., Okazaki K., Kingsbury L., Yoshida K., Müller U., Larue D. T., Winer J. A., Sakano H. Detection of somatic DNA recombination in the transgenic mouse brain. Science. 1991 Oct 4;254(5028):81–86. doi: 10.1126/science.1925563. [DOI] [PubMed] [Google Scholar]
  20. Parker C. N., Halford S. E. Dynamics of long-range interactions on DNA: the speed of synapsis during site-specific recombination by resolvase. Cell. 1991 Aug 23;66(4):781–791. doi: 10.1016/0092-8674(91)90121-e. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Reed R. R., Moser C. D. Resolvase-mediated recombination intermediates contain a serine residue covalently linked to DNA. Cold Spring Harb Symp Quant Biol. 1984;49:245–249. doi: 10.1101/sqb.1984.049.01.028. [DOI] [PubMed] [Google Scholar]
  24. Salvo J. J., Grindley N. D. The gamma delta resolvase bends the res site into a recombinogenic complex. EMBO J. 1988 Nov;7(11):3609–3616. doi: 10.1002/j.1460-2075.1988.tb03239.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Spengler S. J., Stasiak A., Cozzarelli N. R. The stereostructure of knots and catenanes produced by phage lambda integrative recombination: implications for mechanism and DNA structure. Cell. 1985 Aug;42(1):325–334. doi: 10.1016/s0092-8674(85)80128-8. [DOI] [PubMed] [Google Scholar]
  27. Stark W. M., Boocock M. R., Sherratt D. J. Site-specific recombination by Tn3 resolvase. Trends Genet. 1989 Sep;5(9):304–309. doi: 10.1016/0168-9525(89)90113-3. [DOI] [PubMed] [Google Scholar]
  28. Stark W. M., Grindley N. D., Hatfull G. F., Boocock M. R. Resolvase-catalysed reactions between res sites differing in the central dinucleotide of subsite I. EMBO J. 1991 Nov;10(11):3541–3548. doi: 10.1002/j.1460-2075.1991.tb04918.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Stark W. M., Sherratt D. J., Boocock M. R. Site-specific recombination by Tn3 resolvase: topological changes in the forward and reverse reactions. Cell. 1989 Aug 25;58(4):779–790. doi: 10.1016/0092-8674(89)90111-6. [DOI] [PubMed] [Google Scholar]
  30. Sundin O., Varshavsky A. Arrest of segregation leads to accumulation of highly intertwined catenated dimers: dissection of the final stages of SV40 DNA replication. Cell. 1981 Sep;25(3):659–669. doi: 10.1016/0092-8674(81)90173-2. [DOI] [PubMed] [Google Scholar]
  31. Tonegawa S. Somatic generation of antibody diversity. Nature. 1983 Apr 14;302(5909):575–581. doi: 10.1038/302575a0. [DOI] [PubMed] [Google Scholar]
  32. Volkert F. C., Broach J. R. Site-specific recombination promotes plasmid amplification in yeast. Cell. 1986 Aug 15;46(4):541–550. doi: 10.1016/0092-8674(86)90879-2. [DOI] [PubMed] [Google Scholar]
  33. Wasserman S. A., Cozzarelli N. R. Biochemical topology: applications to DNA recombination and replication. Science. 1986 May 23;232(4753):951–960. doi: 10.1126/science.3010458. [DOI] [PubMed] [Google Scholar]
  34. Wasserman S. A., Cozzarelli N. R. Determination of the stereostructure of the product of Tn3 resolvase by a general method. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1079–1083. doi: 10.1073/pnas.82.4.1079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wasserman S. A., Dungan J. M., Cozzarelli N. R. Discovery of a predicted DNA knot substantiates a model for site-specific recombination. Science. 1985 Jul 12;229(4709):171–174. doi: 10.1126/science.2990045. [DOI] [PubMed] [Google Scholar]

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