Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1997 May 1;16(9):2528–2534. doi: 10.1093/emboj/16.9.2528

Near-simultaneous DNA cleavage by the subunits of the junction-resolving enzyme T4 endonuclease VII.

M J Giraud-Panis 1, D M Lilley 1
PMCID: PMC1169852  PMID: 9171365

Abstract

In common with a number of other DNA junction-resolving enzymes, endonuclease VII of bacteriophage T4 binds to a four-way DNA junction as a dimer, and cleaves two strands of the junction. We have used a supercoil-stabilized cruciform substrate to probe the simultaneity of cleavage at the two sites. Active endonuclease VII converts the supercoiled circular DNA directly into linear product, indicating that the two cleavage reactions must occur within the lifetime of the protein-junction complex. By contrast, a heterodimer of active enzyme and an inactive mutant endonuclease VII leads to the formation of nicked circular product, showing that the subunits operate fully independently.

Full Text

The Full Text of this article is available as a PDF (305.9 KB).

Selected References

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

  1. Ariyoshi M., Vassylyev D. G., Iwasaki H., Nakamura H., Shinagawa H., Morikawa K. Atomic structure of the RuvC resolvase: a holliday junction-specific endonuclease from E. coli. Cell. 1994 Sep 23;78(6):1063–1072. doi: 10.1016/0092-8674(94)90280-1. [DOI] [PubMed] [Google Scholar]
  2. Bennett R. J., Dunderdale H. J., West S. C. Resolution of Holliday junctions by RuvC resolvase: cleavage specificity and DNA distortion. Cell. 1993 Sep 24;74(6):1021–1031. doi: 10.1016/0092-8674(93)90724-5. [DOI] [PubMed] [Google Scholar]
  3. Bennett R. J., West S. C. RuvC protein resolves Holliday junctions via cleavage of the continuous (noncrossover) strands. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5635–5639. doi: 10.1073/pnas.92.12.5635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Elborough K. M., West S. C. Resolution of synthetic Holliday junctions in DNA by an endonuclease activity from calf thymus. EMBO J. 1990 Sep;9(9):2931–2936. doi: 10.1002/j.1460-2075.1990.tb07484.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gellert M., Mizuuchi K., O'Dea M. H., Ohmori H., Tomizawa J. DNA gyrase and DNA supercoiling. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):35–40. doi: 10.1101/sqb.1979.043.01.007. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. 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]
  9. Lilley D. M., Hallam L. R. Thermodynamics of the ColE1 cruciform. Comparisons between probing and topological experiments using single topoisomers. J Mol Biol. 1984 Nov 25;180(1):179–200. doi: 10.1016/0022-2836(84)90436-4. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. McCulloch R., Coggins L. W., Colloms S. D., Sherratt D. J. Xer-mediated site-specific recombination at cer generates Holliday junctions in vivo. EMBO J. 1994 Apr 15;13(8):1844–1855. doi: 10.1002/j.1460-2075.1994.tb06453.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Mueller J. E., Kemper B., Cunningham R. P., Kallenbach N. R., Seeman N. C. T4 endonuclease VII cleaves the crossover strands of Holliday junction analogs. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9441–9445. doi: 10.1073/pnas.85.24.9441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Mueller J. E., Newton C. J., Jensch F., Kemper B., Cunningham R. P., Kallenbach N. R., Seeman N. C. Resolution of Holliday junction analogs by T4 endonuclease VII can be directed by substrate structure. J Biol Chem. 1990 Aug 15;265(23):13918–13924. [PubMed] [Google Scholar]
  15. Murchie A. I., Lilley D. M. Supercoiled DNA and cruciform structures. Methods Enzymol. 1992;211:158–180. doi: 10.1016/0076-6879(92)11010-g. [DOI] [PubMed] [Google Scholar]
  16. Pottmeyer S., Kemper B. T4 endonuclease VII resolves cruciform DNA with nick and counter-nick and its activity is directed by local nucleotide sequence. J Mol Biol. 1992 Feb 5;223(3):607–615. doi: 10.1016/0022-2836(92)90977-r. [DOI] [PubMed] [Google Scholar]
  17. Pöhler J. R., Giraud-Panis M. J., Lilley D. M. T4 endonuclease VII selects and alters the structure of the four-way DNA junction; binding of a resolution-defective mutant enzyme. J Mol Biol. 1996 Aug 2;260(5):678–696. doi: 10.1006/jmbi.1996.0430. [DOI] [PubMed] [Google Scholar]
  18. Saito A., Iwasaki H., Ariyoshi M., Morikawa K., Shinagawa H. Identification of four acidic amino acids that constitute the catalytic center of the RuvC Holliday junction resolvase. Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7470–7474. doi: 10.1073/pnas.92.16.7470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. White M. F., Lilley D. M. The structure-selectivity and sequence-preference of the junction-resolving enzyme CCE1 of Saccharomyces cerevisiae. J Mol Biol. 1996 Mar 29;257(2):330–341. doi: 10.1006/jmbi.1996.0166. [DOI] [PubMed] [Google Scholar]

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

RESOURCES