Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1998 Sep 1;26(17):4012–4018. doi: 10.1093/nar/26.17.4012

The role of supercoiling in mycobacteriophage L5 integrative recombination.

C E Peña 1, J M Kahlenberg 1, G F Hatfull 1
PMCID: PMC147811  PMID: 9705513

Abstract

The genome of temperate mycobacteriophage L5 integrates into the chromosomes of its hosts, including Mycobacterium smegmatis , Mycobacterium tuberculosis and bacille Calmette-Guérin. This integrase-mediated site-specific recombination reaction occurs between the phage attP site and the mycobacterial attB site and requires the mycobacterial integration host factor. Here we examine the role of supercoiling in this reaction and show that integration is stimulated by DNA supercoiling but that supercoiling of either the attP or the attB substrate enhances recombination. Supercoiling thus facilitates a post-synaptic recombination event. We also show that, while supercoiling is not required for the production of a recombinagenic intasome, a mutant attP DNA deficient in binding of the host factor acquires a dependence on supercoiling for intasome formation and recombination.

Full Text

The Full Text of this article is available as a PDF (317.6 KB).

Selected References

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

  1. Abremski K., Gottesman S. The form of the DNA substrate required for excisive recombination of bacteriophage lambda. J Mol Biol. 1979 Jul 5;131(3):637–649. doi: 10.1016/0022-2836(79)90012-3. [DOI] [PubMed] [Google Scholar]
  2. Abremski K., Hoess R., Sternberg N. Studies on the properties of P1 site-specific recombination: evidence for topologically unlinked products following recombination. Cell. 1983 Apr;32(4):1301–1311. doi: 10.1016/0092-8674(83)90311-2. [DOI] [PubMed] [Google Scholar]
  3. Benjamin K. R., Abola A. P., Kanaar R., Cozzarelli N. R. Contributions of supercoiling to Tn3 resolvase and phage Mu Gin site-specific recombination. J Mol Biol. 1996 Feb 16;256(1):50–65. doi: 10.1006/jmbi.1996.0067. [DOI] [PubMed] [Google Scholar]
  4. Ford M. E., Sarkis G. J., Belanger A. E., Hendrix R. W., Hatfull G. F. Genome structure of mycobacteriophage D29: implications for phage evolution. J Mol Biol. 1998 May 29;279(1):143–164. doi: 10.1006/jmbi.1997.1610. [DOI] [PubMed] [Google Scholar]
  5. Griffith J. D., Nash H. A. Genetic rearrangement of DNA induces knots with a unique topology: implications for the mechanism of synapsis and crossing-over. Proc Natl Acad Sci U S A. 1985 May;82(10):3124–3128. doi: 10.1073/pnas.82.10.3124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Guo F., Gopaul D. N., van Duyne G. D. Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse. Nature. 1997 Sep 4;389(6646):40–46. doi: 10.1038/37925. [DOI] [PubMed] [Google Scholar]
  7. Hakimi J. M., Scocca J. J. Purification and characterization of the integrase from the Haemophilus influenzae bacteriophage HP1; identification of a four-stranded intermediate and the order of strand exchange. Mol Microbiol. 1996 Jul;21(1):147–158. doi: 10.1046/j.1365-2958.1996.6311351.x. [DOI] [PubMed] [Google Scholar]
  8. Hatfull G. F., Sarkis G. J. DNA sequence, structure and gene expression of mycobacteriophage L5: a phage system for mycobacterial genetics. Mol Microbiol. 1993 Feb;7(3):395–405. doi: 10.1111/j.1365-2958.1993.tb01131.x. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Kikuchi Y., Nash H. A. Nicking-closing activity associated with bacteriophage lambda int gene product. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3760–3764. doi: 10.1073/pnas.76.8.3760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kikuchi Y., Nash H. Integrative recombination of bacteriophage lambda: requirement for supertwisted DNA in vivo and characterization of int. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 2):1099–1109. doi: 10.1101/sqb.1979.043.01.122. [DOI] [PubMed] [Google Scholar]
  12. Kwon H. J., Tirumalai R., Landy A., Ellenberger T. Flexibility in DNA recombination: structure of the lambda integrase catalytic core. Science. 1997 Apr 4;276(5309):126–131. doi: 10.1126/science.276.5309.126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Lee M. H., Hatfull G. F. Mycobacteriophage L5 integrase-mediated site-specific integration in vitro. J Bacteriol. 1993 Nov;175(21):6836–6841. doi: 10.1128/jb.175.21.6836-6841.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lee M. H., Pascopella L., Jacobs W. R., Jr, Hatfull G. F. Site-specific integration of mycobacteriophage L5: integration-proficient vectors for Mycobacterium smegmatis, Mycobacterium tuberculosis, and bacille Calmette-Guérin. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3111–3115. doi: 10.1073/pnas.88.8.3111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mizuuchi K., Gellert M., Nash H. A. Involement of supertwisted DNA in integrative recombination of bacteriophage lambda. J Mol Biol. 1978 May 25;121(3):375–392. doi: 10.1016/0022-2836(78)90370-4. [DOI] [PubMed] [Google Scholar]
  17. Mizuuchi K., Mizuuchi M. Integrative recombination of bacteriophage lambda: in vitro study of the intermolecular reaction. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 2):1111–1114. doi: 10.1101/sqb.1979.043.01.123. [DOI] [PubMed] [Google Scholar]
  18. Pedulla M. L., Lee M. H., Lever D. C., Hatfull G. F. A novel host factor for integration of mycobacteriophage L5. Proc Natl Acad Sci U S A. 1996 Dec 24;93(26):15411–15416. doi: 10.1073/pnas.93.26.15411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Peña C. E., Lee M. H., Pedulla M. L., Hatfull G. F. Characterization of the mycobacteriophage L5 attachment site, attP. J Mol Biol. 1997 Feb 14;266(1):76–92. doi: 10.1006/jmbi.1996.0774. [DOI] [PubMed] [Google Scholar]
  20. Peña C. E., Stoner J. E., Hatfull G. F. Positions of strand exchange in mycobacteriophage L5 integration and characterization of the attB site. J Bacteriol. 1996 Sep;178(18):5533–5536. doi: 10.1128/jb.178.18.5533-5536.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pollock T. J., Abremski K. DNA without supertwists can be an in vitro substrate for site-specific recombination of bacteriophage lambda. J Mol Biol. 1979 Jul 5;131(3):651–654. doi: 10.1016/0022-2836(79)90013-5. [DOI] [PubMed] [Google Scholar]
  22. Pollock T. J., Nash H. A. Knotting of DNA caused by a genetic rearrangement. Evidence for a nucleosome-like structure in site-specific recombination of bacteriophage lambda. J Mol Biol. 1983 Oct 15;170(1):1–18. doi: 10.1016/s0022-2836(83)80224-1. [DOI] [PubMed] [Google Scholar]
  23. Richet E., Abcarian P., Nash H. A. The interaction of recombination proteins with supercoiled DNA: defining the role of supercoiling in lambda integrative recombination. Cell. 1986 Sep 26;46(7):1011–1021. doi: 10.1016/0092-8674(86)90700-2. [DOI] [PubMed] [Google Scholar]
  24. Sadowski P. D. Site-specific genetic recombination: hops, flips, and flops. FASEB J. 1993 Jun;7(9):760–767. doi: 10.1096/fasebj.7.9.8392474. [DOI] [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. Snapper S. B., Lugosi L., Jekkel A., Melton R. E., Kieser T., Bloom B. R., Jacobs W. R., Jr Lysogeny and transformation in mycobacteria: stable expression of foreign genes. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6987–6991. doi: 10.1073/pnas.85.18.6987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Snapper S. B., Melton R. E., Mustafa S., Kieser T., Jacobs W. R., Jr Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol Microbiol. 1990 Nov;4(11):1911–1919. doi: 10.1111/j.1365-2958.1990.tb02040.x. [DOI] [PubMed] [Google Scholar]
  28. Stark W. M., Boocock M. R., Sherratt D. J. Catalysis by site-specific recombinases. Trends Genet. 1992 Dec;8(12):432–439. [PubMed] [Google Scholar]
  29. Yang W., Steitz T. A. Crystal structure of the site-specific recombinase gamma delta resolvase complexed with a 34 bp cleavage site. Cell. 1995 Jul 28;82(2):193–207. doi: 10.1016/0092-8674(95)90307-0. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES