Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1990 Jul;87(14):5336–5340. doi: 10.1073/pnas.87.14.5336

The two functional domains of gamma delta resolvase act on the same recombination site: implications for the mechanism of strand exchange.

P Dröge 1, G F Hatfull 1, N D Grindley 1, N R Cozzarelli 1
PMCID: PMC54318  PMID: 2164677

Abstract

During site-specific recombination by the gamma delta resolvase, four DNA strands are broken, exchanged, and religated. This exchange is carried out within a DNA-protein complex, the synaptosome, in which the recombination sites, res, are aligned. The domain of resolvase that binds to a res site is distinct from the domain that breaks and rejoins the DNA. We tested whether the catalytic domain acts on the res site to which its binding domain is bound (in cis) or on the opposing res site in the synaptic complex (in trans). We constructed a hybrid synaptosome in which one res site is bound to wild-type resolvase and the other is bound to a mutant resolvase that binds normally but is unable to break DNA. From the pattern of strand breakage in the reaction intermediate containing resolvase covalently attached to DNA, we conclude that resolvase attacks predominantly, if not exclusively, in cis. Because cis breakage and reunion per se cannot lead to recombination, our results support a model in which DNA exchange is guided by an exchange of resolvase subunits between the breakage and reunion events.

Full text

PDF
5336

Images in this article

5337Image
on p.5337
5337Image
on p.5337
5337Image
on p.5337
5338Image
on p.5338
5339Image
on p.5339
5339Image
on p.5339
5339Image
on p.5339

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. 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]
  3. 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]
  4. 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]
  5. Better M., Lu C., Williams R. C., Echols H. Site-specific DNA condensation and pairing mediated by the int protein of bacteriophage lambda. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5837–5841. doi: 10.1073/pnas.79.19.5837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Falvey E., Hatfull G. F., Grindley N. D. Uncoupling of the recombination and topoisomerase activities of the gamma delta resolvase by a mutation at the crossover point. Nature. 1988 Apr 28;332(6167):861–863. doi: 10.1038/332861a0. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. 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]
  11. Jencks W. P. Binding energy, specificity, and enzymic catalysis: the circe effect. Adv Enzymol Relat Areas Mol Biol. 1975;43:219–410. doi: 10.1002/9780470122884.ch4. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. 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]
  15. 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]
  16. Nash H. A. Integration and excision of bacteriophage lambda: the mechanism of conservation site specific recombination. Annu Rev Genet. 1981;15:143–167. doi: 10.1146/annurev.ge.15.120181.001043. [DOI] [PubMed] [Google Scholar]
  17. Newman B. J., Grindley N. D. Mutants of the gamma delta resolvase: a genetic analysis of the recombination function. Cell. 1984 Sep;38(2):463–469. doi: 10.1016/0092-8674(84)90501-4. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Richet E., Abcarian P., Nash H. A. Synapsis of attachment sites during lambda integrative recombination involves capture of a naked DNA by a protein-DNA complex. Cell. 1988 Jan 15;52(1):9–17. doi: 10.1016/0092-8674(88)90526-0. [DOI] [PubMed] [Google Scholar]
  21. Rimphanitchayakit V., Hatfull G. F., Grindley N. D. The 43 residue DNA binding domain of gamma delta resolvase binds adjacent major and minor grooves of DNA. Nucleic Acids Res. 1989 Feb 11;17(3):1035–1050. doi: 10.1093/nar/17.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Wasserman S. A., White J. H., Cozzarelli N. R. The helical repeat of double-stranded DNA varies as a function of catenation and supercoiling. Nature. 1988 Aug 4;334(6181):448–450. doi: 10.1038/334448a0. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES