Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1994 Jun 1;13(11):2714–2724. doi: 10.1002/j.1460-2075.1994.tb06562.x

Dissecting the resolution reaction of lambda integrase using suicide Holliday junction substrates.

S H Kho 1, A Landy 1
PMCID: PMC395146  PMID: 8013469

Abstract

A reciprocal strand exchange between two DNA helices generates the crossed-strand intermediate, or Holliday junction, which is common to many pathways of homologous and site-specific recombination. The Int family of recombinases are unique in their ability to both make and resolve Holliday junctions. Previous experiments utilizing 'synthetic' att site Holliday junctions to study the mechanisms associated with the cleavage, transfer and ligation of DNA strands have been confined to studying reciprocal strand exchanges (a pair of temporally overlapping strand cleavages). To circumvent this limitation, we have designed synthetic suicide Holliday junctions that make it possible to monitor individual DNA strand cleavage events. These substrates contain a pre-existing nick in the vicinity of the Int binding site; when Int introduces a second nick into these substrates, the 5'OH nucleophile required for ligation (in either the forward or reverse reaction) is lost by diffusion, thus trapping the covalent protein-DNA intermediate. The results indicate that resolution (involving two partner Ints) is stimulated by additional 'cross-core' Ints as a result of enhanced cleavage rates, and not as a result of enhanced co-ordination of cleavage. Several models for the role of the 'cross-core' Ints during resolution are discussed, as well as the usefulness of these substrates for studying additional aspects of the Holliday junction resolution reaction.

Full text

PDF

Images in this article

2717Image
on p.2717
2718Image
on p.2718

Selected References

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

  1. Argos P., Landy A., Abremski K., Egan J. B., Haggard-Ljungquist E., Hoess R. H., Kahn M. L., Kalionis B., Narayana S. V., Pierson L. S., 3rd The integrase family of site-specific recombinases: regional similarities and global diversity. EMBO J. 1986 Feb;5(2):433–440. doi: 10.1002/j.1460-2075.1986.tb04229.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bauer C. E., Gardner J. F., Gumport R. I. Extent of sequence homology required for bacteriophage lambda site-specific recombination. J Mol Biol. 1985 Jan 20;181(2):187–197. doi: 10.1016/0022-2836(85)90084-1. [DOI] [PubMed] [Google Scholar]
  3. Broker T. R., Lehman I. R. Branched DNA molecules: intermediates in T4 recombination. J Mol Biol. 1971 Aug 28;60(1):131–149. doi: 10.1016/0022-2836(71)90453-0. [DOI] [PubMed] [Google Scholar]
  4. Bushman W., Yin S., Thio L. L., Landy A. Determinants of directionality in lambda site-specific recombination. Cell. 1984 Dec;39(3 Pt 2):699–706. doi: 10.1016/0092-8674(84)90477-x. [DOI] [PubMed] [Google Scholar]
  5. Chen J. W., Lee J., Jayaram M. DNA cleavage in trans by the active site tyrosine during Flp recombination: switching protein partners before exchanging strands. Cell. 1992 May 15;69(4):647–658. doi: 10.1016/0092-8674(92)90228-5. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Cooper J. P., Hagerman P. J. Geometry of a branched DNA structure in solution. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7336–7340. doi: 10.1073/pnas.86.19.7336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Craig N. L., Nash H. A. E. coli integration host factor binds to specific sites in DNA. Cell. 1984 Dec;39(3 Pt 2):707–716. doi: 10.1016/0092-8674(84)90478-1. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Craig N. L. The mechanism of conservative site-specific recombination. Annu Rev Genet. 1988;22:77–105. doi: 10.1146/annurev.ge.22.120188.000453. [DOI] [PubMed] [Google Scholar]
  12. Dickie P., McFadden G., Morgan A. R. The site-specific cleavage of synthetic Holliday junction analogs and related branched DNA structures by bacteriophage T7 endonuclease I. J Biol Chem. 1987 Oct 25;262(30):14826–14836. [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. Franz B., Landy A. Interactions between lambda Int molecules bound to sites in the region of strand exchange are required for efficient Holliday junction resolution. J Mol Biol. 1990 Oct 20;215(4):523–535. doi: 10.1016/s0022-2836(05)80165-2. [DOI] [PubMed] [Google Scholar]
  17. Han Y. W., Gumport R. I., Gardner J. F. Complementation of bacteriophage lambda integrase mutants: evidence for an intersubunit active site. EMBO J. 1993 Dec;12(12):4577–4584. doi: 10.1002/j.1460-2075.1993.tb06146.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Hsu P. L., Landy A. Resolution of synthetic att-site Holliday structures by the integrase protein of bacteriophage lambda. Nature. 1984 Oct 25;311(5988):721–726. doi: 10.1038/311721a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. 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]
  23. Kulpa J., Dixon J. E., Pan G., Sadowski P. D. Mutations of the FLP recombinase gene that cause a deficiency in DNA bending and strand cleavage. J Biol Chem. 1993 Jan 15;268(2):1101–1108. [PubMed] [Google Scholar]
  24. 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]
  25. Landy A., Ross W. Viral integration and excision: structure of the lambda att sites. Science. 1977 Sep 16;197(4309):1147–1160. doi: 10.1126/science.331474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lilley D. M., Kemper B. Cruciform-resolvase interactions in supercoiled DNA. Cell. 1984 Feb;36(2):413–422. doi: 10.1016/0092-8674(84)90234-4. [DOI] [PubMed] [Google Scholar]
  27. Lu M., Guo Q., Marky L. A., Seeman N. C., Kallenbach N. R. Thermodynamics of DNA branching. J Mol Biol. 1992 Feb 5;223(3):781–789. doi: 10.1016/0022-2836(92)90989-w. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. 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]
  31. Mizuuchi K., Adzuma K. Inversion of the phosphate chirality at the target site of Mu DNA strand transfer: evidence for a one-step transesterification mechanism. Cell. 1991 Jul 12;66(1):129–140. doi: 10.1016/0092-8674(91)90145-o. [DOI] [PubMed] [Google Scholar]
  32. Mizuuchi K., Kemper B., Hays J., Weisberg R. A. T4 endonuclease VII cleaves holliday structures. Cell. 1982 Jun;29(2):357–365. doi: 10.1016/0092-8674(82)90152-0. [DOI] [PubMed] [Google Scholar]
  33. Mizuuchi K., Weisberg R., Enquist L., Mizuuchi M., Buraczynska M., Foeller C., Hsu P. L., Ross W., Landy A. Structure and function of the phage lambda att site: size, int-binding sites, and location of the crossover point. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):429–437. doi: 10.1101/sqb.1981.045.01.057. [DOI] [PubMed] [Google Scholar]
  34. Moitoso de Vargas L., Pargellis C. A., Hasan N. M., Bushman E. W., Landy A. Autonomous DNA binding domains of lambda integrase recognize two different sequence families. Cell. 1988 Sep 23;54(7):923–929. doi: 10.1016/0092-8674(88)90107-9. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Murchie A. I., Portugal J., Lilley D. M. Cleavage of a four-way DNA junction by a restriction enzyme spanning the point of strand exchange. EMBO J. 1991 Mar;10(3):713–718. doi: 10.1002/j.1460-2075.1991.tb08001.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nunes-Düby S. E., Matsumoto L., Landy A. Half-att site substrates reveal the homology independence and minimal protein requirements for productive synapsis in lambda excisive recombination. Cell. 1989 Oct 6;59(1):197–206. doi: 10.1016/0092-8674(89)90881-7. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. Pan G., Sadowski P. D. Ligation activity of FLP recombinase. The strand ligation activity of a site-specific recombinase using an activated DNA substrate. J Biol Chem. 1992 Jun 25;267(18):12397–12399. [PubMed] [Google Scholar]
  41. Pargellis C. A., Nunes-Düby S. E., de Vargas L. M., Landy A. Suicide recombination substrates yield covalent lambda integrase-DNA complexes and lead to identification of the active site tyrosine. J Biol Chem. 1988 Jun 5;263(16):7678–7685. [PubMed] [Google Scholar]
  42. Ross W., Landy A., Kikuchi Y., Nash H. Interaction of int protein with specific sites on lambda att DNA. Cell. 1979 Oct;18(2):297–307. doi: 10.1016/0092-8674(79)90049-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schwartz C. J., Sadowski P. D. FLP protein of 2 mu circle plasmid of yeast induces multiple bends in the FLP recognition target site. J Mol Biol. 1990 Nov 20;216(2):289–298. doi: 10.1016/s0022-2836(05)80320-1. [DOI] [PubMed] [Google Scholar]
  44. Schwartz C. J., Sadowski P. D. FLP recombinase of the 2 microns circle plasmid of Saccharomyces cerevisiae bends its DNA target. Isolation of FLP mutants defective in DNA bending. J Mol Biol. 1989 Feb 20;205(4):647–658. doi: 10.1016/0022-2836(89)90310-0. [DOI] [PubMed] [Google Scholar]
  45. Sigal N., Alberts B. Genetic recombination: the nature of a crossed strand-exchange between two homologous DNA molecules. J Mol Biol. 1972 Nov 28;71(3):789–793. doi: 10.1016/s0022-2836(72)80039-1. [DOI] [PubMed] [Google Scholar]
  46. Smith G. R. Homologous recombination in procaryotes. Microbiol Rev. 1988 Mar;52(1):1–28. doi: 10.1128/mr.52.1.1-28.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sobell H. M. Molecular mechanism for genetic recombination. Proc Natl Acad Sci U S A. 1972 Sep;69(9):2483–2487. doi: 10.1073/pnas.69.9.2483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. Trask D. K., DiDonato J. A., Muller M. T. Rapid detection and isolation of covalent DNA/protein complexes: application to topoisomerase I and II. EMBO J. 1984 Mar;3(3):671–676. doi: 10.1002/j.1460-2075.1984.tb01865.x. [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. West S. C. Enzymes and molecular mechanisms of genetic recombination. Annu Rev Biochem. 1992;61:603–640. doi: 10.1146/annurev.bi.61.070192.003131. [DOI] [PubMed] [Google Scholar]
  52. Yin S., Bushman W., Landy A. Interaction of the lambda site-specific recombination protein Xis with attachment site DNA. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1040–1044. doi: 10.1073/pnas.82.4.1040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. 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]
  54. de Massy B., Studier F. W., Dorgai L., Appelbaum E., Weisberg R. A. Enzymes and sites of genetic recombination: studies with gene-3 endonuclease of phage T7 and with site-affinity mutants of phage lambda. Cold Spring Harb Symp Quant Biol. 1984;49:715–726. doi: 10.1101/sqb.1984.049.01.081. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES