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Structure-Function Studies of Bacteriophage P2 Integrase and Cox Protein

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Structure-Function Studies of Bacteriophage P2 Integrase and Cox Protein Structure-Function Studies of Bacteriophage P2 Integrase and Cox Protein A Detailed Study of Two Proteins Involved in the Choice of Bacteriophage Reproductive Mode Jesper Eriksson Department of Genetics, Microbiology and Toxicology Stockholm University Stockholm 2005 1 Abstract Probably no group of organisms has been as important as bacteriophages when it comes to the understanding of fundamental biological processes like transcriptional control, DNA replication, site-specific recombination, e.t.c. The work presented in this thesis is a contribution towards the complete understanding of these organisms. Two proteins, integrase, and Cox, which are important for the choice of the life mode of bacteriophage P2, are inves- tigated. P2 is a temperate phage, i.e. it can either insert its DNA into the host chromosome (by site-specific recombination) and wait (lysogeny), or it can produce new progeny with the help of the host protein machinery and there- after lyse the cell (lytic cycle). The integrase protein is necessary for the integration and excision of the phage genome. The Cox protein is involved as a directional factor in the site-specific recombination, where it stimulates excision and inhibits integration. It has been shown that the Cox protein also is important for the choice of the lytic cycle. The choice of life mode is regu- lated on a transcriptional level, where two mutually exclusive promoters direct whether the lytic cycle (Pe) or lysogeny (Pc) is chosen. The Cox pro- tein has been shown to repress the Pc promoter and thereby making tran- scription from the Pe promoter possible, leading to the lytic cycle. Further, the Cox protein can function as a transcriptional activator on the parasite phage, P4. P4 has gained the ability to adopt the P2 protein machinery to its own purposes. In this work the importance of the native size for biologically active inte- grase and Cox proteins has been determined. Further, structure-function analyses of the two proteins have been performed with focus on the protein- protein interfaces. In addition it is shown that P2 Cox and the P2 relative WΦ Cox changes the DNA topology upon specific binding. From the ob- tained results a mechanism for P2 Cox-DNA interaction is discussed. The results from this thesis can be used in the development of a gene de- livery system based on the P2 site-specific recombination system. © Jesper Eriksson, Stockholm 2005 ISBN 91-7155-128-X pp1-82 Typesetting: Intellecta Docusys Printed in Sweden by Intellecta Docusys, Stockholm 2005 Distributor: Stockholm University Library 2 To Sara, Moa and Ida 3 Munen Muso Meikyo Shisui -“Clear Mind Reflects Like Quiet Water” Kendo Philosophy 4 List of publications This thesis is based on the following articles as well as unpublished results: I. Eriksson, J.M. and Haggård-Ljungquist, E. 2000. The multifunc- tional bacteriophage Cox protein requires oligomerization for bio- logical activity. Journal of Bacteriology 182:6714-6723. II. Ahlgren-Berg, A., Eriksson, J.M., and Haggård-Ljungquist, E. 2005. A comparative analysis of the multifunctional Cox proteins of the two heteroimmune phages P2 and WΦ. Manuscript. III. Frumerie, C., Eriksson, J.M., Dugast, M., and Haggård- Ljungquist, E. 2005. Dimerization of bacteriophage P2 integrase is not required for binding to its DNA target but for its biological activity. Gene 344: 221-231. The cover illustration is a modified image of the central section of the bacteriophage P2 head, published in “Dokland, T., Lindqvist, B:H: and Fuller, S.D. (1992) EMBO Journal 11:839-846”. 5 6 Contents Abstract ...........................................................................................................2 List of publications...........................................................................................5 Introduction ...................................................................................................11 Background ...................................................................................................13 Bacteriophage P2 .......................................................................................................13 P2 propagation ......................................................................................................15 P2 lytic-lysogeny switch.........................................................................................16 P2 site-specific recombination ...............................................................................17 P2-P4 interaction ...................................................................................................19 P2 Cox protein and its DNA substrates..................................................................21 P2 integrase and its DNA substrates .....................................................................22 P2 Int regulation ....................................................................................................22 WΦ - a P2 relative .................................................................................................23 Conservative site-specific recombination ....................................................................24 Introduction............................................................................................................24 Tyrosine Recombinases ........................................................................................25 Recombination directionality factors (RDFs)..........................................................27 The mechanism of conservative site-specific recombination..................................29 Regulation of active sites.......................................................................................31 Cis versus trans cleavage......................................................................................32 Intasome formation-putting the pieces together .....................................................32 Regulation of transcriptional initiation..........................................................................37 Introduction............................................................................................................37 The RNA polymerase ............................................................................................37 The promoter element ...........................................................................................37 Initiation of transcription.........................................................................................38 Regulation of transcriptional initiation ....................................................................39 DNA-Bending and transcriptional regulation ..........................................................41 The lysis-lysogeny switch of bacteriophage λ ........................................................42 Present investigation.....................................................................................45 Aim of the study ..........................................................................................................45 Specific Methods.........................................................................................................45 Results and Discussion...............................................................................................46 List and brief summary of the papers.....................................................................46 P2 Cox forms multimers in vivo (Paper I)...............................................................47 P2 Cox forms dimers, trimers, tetramers and octamers in vitro (Paper I and II) .....47 P2 Cox binds cooperatively to DNA (Paper I) ........................................................48 Oligomerization is essential for DNA binding and P2 Cox activity (Paper I) ...........48 The C-terminal part of the P2 Cox protein is involved in oligomerization (Paper I).48 7 Investigation of the WΦ Cox binding sites (Paper II)..............................................49 Comparison of P2 and WΦ Cox in vitro oligomerization (Paper II).........................50 Domain swapping between P2 Cox and WΦ Cox (Paper II) ..................................50 DNA length requirement for specific P2 Cox-DNA interactions (Unpublished) .......50 Specific P2 Cox and WΦ Cox binding induces a large bend in the DNA targets (Paper II)................................................................................................................52 P2 Cox does not bend DNA when binding non-specific substrates (Paper II)........53 P2 integrase forms dimers in vivo, but does not show cooperative binding or oligomerization (Paper III)......................................................................................53 Sequencing of int defective mutants (Unpublished) ...............................................54 Residues affecting Int dimerization are located in the C-terminal part of the protein (Unpublished results).............................................................................................55 The absolute C-terminal end is also involved in dimerization of P2 integrase (Paper III) ..........................................................................................................................56 Residue E197 in P2 integrase is involved in Int dimerization (Paper III) ................56 Capacities of the mutated and truncated Int proteins to complement the int1 defective P2 prophage (Paper III and unpublished results)....................................57 The C-truncated integrase proteins
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