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. 1994 Jan;66(1):99–109. doi: 10.1016/S0006-3495(94)80765-9

The solution structure of the four-way DNA junction at low-salt conditions: a fluorescence resonance energy transfer analysis.

R M Clegg 1, A I Murchie 1, D M Lilley 1
PMCID: PMC1275668  PMID: 8130350

Abstract

The four-way DNA (Holliday) junction is an important postulated intermediate in the process of genetic recombination. Earlier studies have suggested that the junction exists in two alternative conformations, depending upon the salt concentration present. At high salt concentrations the junction folds into a stacked X structure, while at low salt concentrations the data indicate an extended unstacked conformation. The stereochemical conformation of the four-way DNA junction at low salt (low alkali ion concentration and no alkaline earth ions) was established by comparing the efficiency of fluorescence resonance energy transfer (FRET) between donor and acceptor molecules attached pairwise in three permutations to the 5' termini of the duplex arms. A new variation of FRET was implemented based upon a systematic variation of the fraction of donor labeled single strands. The FRET results indicate that the structure of the four-way DNA junction at low salt exists as an unstacked, extended, square arrangement of the four duplex arms. The donor titration measurements made in the presence of magnesium ions clearly show the folding of the junction into the X stacked structure. In addition, the FRET efficiency can be measured. The fluorescence anisotropy of the acceptor in the presence of Mg2+ during donor titrations was also measured; the FRET efficiency can be calculated from the anisotropy data and the results are consistent with the folded, stacked X structure.

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Selected References

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

  1. Cantor C. R., Pechukas P. Determination of distance distribution functions by singlet-singlet energy transfer. Proc Natl Acad Sci U S A. 1971 Sep;68(9):2099–2101. doi: 10.1073/pnas.68.9.2099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cardullo R. A., Agrawal S., Flores C., Zamecnik P. C., Wolf D. E. Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8790–8794. doi: 10.1073/pnas.85.23.8790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Churchill M. E., Tullius T. D., Kallenbach N. R., Seeman N. C. A Holliday recombination intermediate is twofold symmetric. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4653–4656. doi: 10.1073/pnas.85.13.4653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clegg R. M. Fluorescence resonance energy transfer and nucleic acids. Methods Enzymol. 1992;211:353–388. doi: 10.1016/0076-6879(92)11020-j. [DOI] [PubMed] [Google Scholar]
  5. Clegg R. M., Murchie A. I., Zechel A., Carlberg C., Diekmann S., Lilley D. M. Fluorescence resonance energy transfer analysis of the structure of the four-way DNA junction. Biochemistry. 1992 May 26;31(20):4846–4856. doi: 10.1021/bi00135a016. [DOI] [PubMed] [Google Scholar]
  6. Clegg R. M., Murchie A. I., Zechel A., Lilley D. M. Observing the helical geometry of double-stranded DNA in solution by fluorescence resonance energy transfer. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2994–2998. doi: 10.1073/pnas.90.7.2994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Connolly B. A. The synthesis of oligonucleotides containing a primary amino group at the 5'-terminus. Nucleic Acids Res. 1987 Apr 10;15(7):3131–3139. doi: 10.1093/nar/15.7.3131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Conrad R. H., Brand L. Intramolecular transfer of excitation from tryptophan to 1-dimethylaminonaphthalene 5-sulfonamide in a series of model compounds. Biochemistry. 1968 Feb;7(2):777–787. doi: 10.1021/bi00842a036. [DOI] [PubMed] [Google Scholar]
  9. Cooper J. P., Hagerman P. J. Gel electrophoretic analysis of the geometry of a DNA four-way junction. J Mol Biol. 1987 Dec 20;198(4):711–719. doi: 10.1016/0022-2836(87)90212-9. [DOI] [PubMed] [Google Scholar]
  10. De Boeck H., Macgregor R. B., Jr, Clegg R. M., Sharon N., Loontiens F. G. Binding of N-dansylgalactosamine to the lectin from Erythrina cristagalli as followed by stopped-flow and pressure-jump relaxation kinetics. Eur J Biochem. 1985 May 15;149(1):141–145. doi: 10.1111/j.1432-1033.1985.tb08904.x. [DOI] [PubMed] [Google Scholar]
  11. Diekmann S., Pörschke D. Electro-optical analysis of 'curved' DNA fragments. Biophys Chem. 1987 May 9;26(2-3):207–216. doi: 10.1016/0301-4622(87)80023-6. [DOI] [PubMed] [Google Scholar]
  12. Drake J. M., Klafter J., Levitz P. Chemical and biological microstructures as probed by dynamic processes. Science. 1991 Mar 29;251(5001):1574–1579. doi: 10.1126/science.2011737. [DOI] [PubMed] [Google Scholar]
  13. Duckett D. R., Lilley D. M. Effects of base mismatches on the structure of the four-way DNA junction. J Mol Biol. 1991 Sep 5;221(1):147–161. doi: 10.1016/0022-2836(91)80211-c. [DOI] [PubMed] [Google Scholar]
  14. Duckett D. R., Murchie A. I., Diekmann S., von Kitzing E., Kemper B., Lilley D. M. The structure of the Holliday junction, and its resolution. Cell. 1988 Oct 7;55(1):79–89. doi: 10.1016/0092-8674(88)90011-6. [DOI] [PubMed] [Google Scholar]
  15. Duckett D. R., Murchie A. I., Lilley D. M. The role of metal ions in the conformation of the four-way DNA junction. EMBO J. 1990 Feb;9(2):583–590. doi: 10.1002/j.1460-2075.1990.tb08146.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Eisinger J., Feuer B., Lamola A. A. Intramolecular singlet excitation transfer. Applications to polypeptides. Biochemistry. 1969 Oct;8(10):3908–3915. doi: 10.1021/bi00838a005. [DOI] [PubMed] [Google Scholar]
  17. Gough G. W., Lilley D. M. DNA bending induced by cruciform formation. Nature. 1985 Jan 10;313(5998):154–156. doi: 10.1038/313154a0. [DOI] [PubMed] [Google Scholar]
  18. Haas E., Steinberg I. Z. Intramolecular dynamics of chain molecules monitored by fluctuations in efficiency of excitation energy transfer. A theoretical study. Biophys J. 1984 Oct;46(4):429–437. doi: 10.1016/S0006-3495(84)84040-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Haas E., Wilchek M., Katchalski-Katzir E., Steinberg I. Z. Distribution of end-to-end distances of oligopeptides in solution as estimated by energy transfer. Proc Natl Acad Sci U S A. 1975 May;72(5):1807–1811. doi: 10.1073/pnas.72.5.1807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hochstrasser R. A., Chen S. M., Millar D. P. Distance distribution in a dye-linked oligonucleotide determined by time-resolved fluorescence energy transfer. Biophys Chem. 1992 Dec;45(2):133–141. doi: 10.1016/0301-4622(92)87005-4. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. 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]
  24. Kubitscheck U., Kircheis M., Schweitzer-Stenner R., Dreybrodt W., Jovin T. M., Pecht I. Fluorescence resonance energy transfer on single living cells. Application to binding of monovalent haptens to cell-bound immunoglobulin E. Biophys J. 1991 Aug;60(2):307–318. doi: 10.1016/S0006-3495(91)82055-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. McClellan J. A., Lilley D. M. A two-state conformational equilibrium for alternating (A-T)n sequences in negatively supercoiled DNA. J Mol Biol. 1987 Oct 20;197(4):707–721. doi: 10.1016/0022-2836(87)90477-3. [DOI] [PubMed] [Google Scholar]
  26. Meselson M. S., Radding C. M. A general model for genetic recombination. Proc Natl Acad Sci U S A. 1975 Jan;72(1):358–361. doi: 10.1073/pnas.72.1.358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Murchie A. I., Clegg R. M., von Kitzing E., Duckett D. R., Diekmann S., Lilley D. M. Fluorescence energy transfer shows that the four-way DNA junction is a right-handed cross of antiparallel molecules. Nature. 1989 Oct 26;341(6244):763–766. doi: 10.1038/341763a0. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Orr-Weaver T. L., Szostak J. W., Rothstein R. J. Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6354–6358. doi: 10.1073/pnas.78.10.6354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sinha N. D., Biernat J., McManus J., Köster H. Polymer support oligonucleotide synthesis XVIII: use of beta-cyanoethyl-N,N-dialkylamino-/N-morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product. Nucleic Acids Res. 1984 Jun 11;12(11):4539–4557. doi: 10.1093/nar/12.11.4539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stryer L. Fluorescence energy transfer as a spectroscopic ruler. Annu Rev Biochem. 1978;47:819–846. doi: 10.1146/annurev.bi.47.070178.004131. [DOI] [PubMed] [Google Scholar]
  32. Szöllösi J., Trón L., Damjanovich S., Helliwell S. H., Arndt-Jovin D., Jovin T. M. Fluorescence energy transfer measurements on cell surfaces: a critical comparison of steady-state fluorimetric and flow cytometric methods. Cytometry. 1984 Mar;5(2):210–216. doi: 10.1002/cyto.990050216. [DOI] [PubMed] [Google Scholar]
  33. WEBER G. Fluorescence-polarization spectrum and electronic-energy transfer in tyrosine, tryptophan and related compounds. Biochem J. 1960 May;75:335–345. doi: 10.1042/bj0750335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. von Kitzing E., Lilley D. M., Diekmann S. The stereochemistry of a four-way DNA junction: a theoretical study. Nucleic Acids Res. 1990 May 11;18(9):2671–2683. doi: 10.1093/nar/18.9.2671. [DOI] [PMC free article] [PubMed] [Google Scholar]

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