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. 1993 Dec;12(12):4577–4584. doi: 10.1002/j.1460-2075.1993.tb06146.x

Complementation of bacteriophage lambda integrase mutants: evidence for an intersubunit active site.

Y W Han 1, R I Gumport 1, J F Gardner 1
PMCID: PMC413888  PMID: 8223467

Abstract

Site-specific recombination of bacteriophage lambda starts with the formation of higher-order protein--DNA complexes, called 'intasomes', and is followed by a series of steps, including the initial DNA cleavage, top-strand exchange, branch migration and bottom-strand exchange, to produce recombinant products. One of the intasomes formed during excisive recombination (the attL complex) is composed of the phage-encoded integrase (Int), integration host factor (IHF) and one of the recombination substrates, attL DNA. Int is the catalytic recombinase and has two different DNA binding domains. When IHF is present, Int binds to two types of sites in attL DNA, the three arm-type sites (P'123) and the core-type sites (B and C') where the reciprocal strand exchange takes place. The Tyr342 residue of Int serves as a nucleophile during strand cleavage and covalently attaches to the DNA through a phosphotyrosyl bond. In vitro complementation assays have been performed for strand cleavage using attL suicide substrates and mutant proteins containing amino acid substitutions at residues conserved in the integrase family of recombinases. We demonstrate that at least two Int monomers are required to form the catalytically-competent species that performs cleavage at the B site. It is likely that the active site is formed by two Int monomers.

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Selected References

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

  1. Abremski K. E., Hoess R. H. Evidence for a second conserved arginine residue in the integrase family of recombination proteins. Protein Eng. 1992 Jan;5(1):87–91. doi: 10.1093/protein/5.1.87. [DOI] [PubMed] [Google Scholar]
  2. Amin A., Roca H., Luetke K., Sadowski P. D. Synapsis, strand scission, and strand exchange induced by the FLP recombinase: analysis with half-FRT sites. Mol Cell Biol. 1991 Sep;11(9):4497–4508. doi: 10.1128/mcb.11.9.4497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Bauer C. E., Hesse S. D., Gumport R. I., Gardner J. F. Mutational analysis of integrase arm-type binding sites of bacteriophage lambda. Integration and excision involve distinct interactions of integrase with arm-type sites. J Mol Biol. 1986 Dec 5;192(3):513–527. doi: 10.1016/0022-2836(86)90273-1. [DOI] [PubMed] [Google Scholar]
  5. Burgin A. B., Jr, Nash H. A. Symmetry in the mechanism of bacteriophage lambda integrative recombination. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9642–9646. doi: 10.1073/pnas.89.20.9642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Evans B. R., Chen J. W., Parsons R. L., Bauer T. K., Teplow D. B., Jayaram M. Identification of the active site tyrosine of Flp recombinase. Possible relevance of its location to the mechanism of recombination. J Biol Chem. 1990 Oct 25;265(30):18504–18510. [PubMed] [Google Scholar]
  8. Gronostajski R. M., Sadowski P. D. The FLP recombinase of the Saccharomyces cerevisiae 2 microns plasmid attaches covalently to DNA via a phosphotyrosyl linkage. Mol Cell Biol. 1985 Nov;5(11):3274–3279. doi: 10.1128/mcb.5.11.3274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hoess R. H., Abremski K. Mechanism of strand cleavage and exchange in the Cre-lox site-specific recombination system. J Mol Biol. 1985 Feb 5;181(3):351–362. doi: 10.1016/0022-2836(85)90224-4. [DOI] [PubMed] [Google Scholar]
  10. Kim S., Landy A. Lambda Int protein bridges between higher order complexes at two distant chromosomal loci attL and attR. Science. 1992 Apr 10;256(5054):198–203. doi: 10.1126/science.1533056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kim S., Moitoso de Vargas L., Nunes-Düby S. E., Landy A. Mapping of a higher order protein-DNA complex: two kinds of long-range interactions in lambda attL. Cell. 1990 Nov 16;63(4):773–781. doi: 10.1016/0092-8674(90)90143-3. [DOI] [PubMed] [Google Scholar]
  12. Kitts P. A., Nash H. A. An intermediate in the phage lambda site-specific recombination reaction is revealed by phosphorothioate substitution in DNA. Nucleic Acids Res. 1988 Jul 25;16(14B):6839–6856. doi: 10.1093/nar/16.14.6839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Moitoso de Vargas L., Kim S., Landy A. DNA looping generated by DNA bending protein IHF and the two domains of lambda integrase. Science. 1989 Jun 23;244(4911):1457–1461. doi: 10.1126/science.2544029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Numrych T. E., Gumport R. I., Gardner J. F. A comparison of the effects of single-base and triple-base changes in the integrase arm-type binding sites on the site-specific recombination of bacteriophage lambda. Nucleic Acids Res. 1990 Jul 11;18(13):3953–3959. doi: 10.1093/nar/18.13.3953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Qian X. H., Inman R. B., Cox M. M. Reactions between half- and full-FLP recombination target sites. A model system for analyzing early steps in FLP protein-mediated site-specific recombination. J Biol Chem. 1992 Apr 15;267(11):7794–7805. [PubMed] [Google Scholar]
  20. Ross W., Landy A. Bacteriophage lambda int protein recognizes two classes of sequence in the phage att site: characterization of arm-type sites. Proc Natl Acad Sci U S A. 1982 Dec;79(24):7724–7728. doi: 10.1073/pnas.79.24.7724. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ross W., Landy A. Patterns of lambda Int recognition in the regions of strand exchange. Cell. 1983 May;33(1):261–272. doi: 10.1016/0092-8674(83)90355-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Winoto A., Chung S., Abraham J., Echols H. Directional control of site-specific recombination by bacteriophage lambda. Evidence that a binding site for Int protein far from the crossover point is required for integrative but not excisive recombination. J Mol Biol. 1986 Dec 5;192(3):677–680. doi: 10.1016/0022-2836(86)90286-x. [DOI] [PubMed] [Google Scholar]

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