<p>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.</p><p>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 investigated. 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 thereafter 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 regulated 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.</p><p>In this work the importance of the native size for biologically active integrase 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 Wphi Cox changes the DNA topology upon specific binding. From the obtained results a mechanism for P2 Cox-DNA interaction is discussed.</p><p>The results from this thesis can be used in the development of a gene delivery system based on the P2 site-specific recombination system.</p>

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 investigated. 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 thereafter 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 regulated 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 integrase 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 Wphi Cox changes the DNA topology upon specific binding. From the obtained results a mechanism for P2 Cox-DNA interaction is discussed. The results from this thesis can be used in the development of a gene delivery system based on the P2 site-specific recombination system.

Herewithin, two transcription factor (TF) regulatory complexes were investigated. A bHLH–MYB–WDR (BMW) DNA-binding complex from maize was the first complex to be studied. R, a maize bHLH involved in the activation of genes in the anthocyanin pathway, had been characterized to indirectly bind DNA despite the presence of a functional DNA-binding domain. Findings presented here reveal that this is only partially correct. Direct DNA-binding by R was found to be dependent upon two distinct dimerization domains that function as a switch. This switch-like mechanism allows R to be repurposed for the activation of promoters of differing cis-element structure. The second regulatory complex studied was of the Arabidopsis thaliana MIKC-MADS TF family. For many TFs, DNA-binding site recognition is relatively straightforward and very sequence specific, while others exhibit relaxed sequence specificity. MADS-domain TFs are one family of TFs with a wider range of cis-element sequences. Though consensus cis-element sequences have been determined for various MADS-domains, correctly predicting and identifying biologically functional cis-elements has been a challenge. In order to study the influence of nucleobase associations within the cis-element, a DNA-Protein Interaction (DPI)-ELISA method was modified and optimized to screen a panel of specific probes. Screening of the SEP3 homodimer against a panel of sequential, palindromic probes revealed that nucleobases in position -1:+1 of the CArG-box influence binding strength between the MADS-domain and DNA. Additionally, the specificity of AGL15 towards CT-W6-AG forms was discovered to be determined by the functional groups present in the minor groove at position -4:+4 using inosine:cytosine (I:C) base pairs. Finally, the FLC–SVP MADS-domain heterodimer, bound to a native cis-element, was modeled and binding simulated using molecular dynamics. In conjunction with simulations of AGL15 and SEP3 homodimers, a potential binding mechanism was identified for this unique heterodimer. DNA sequence recognition by the MADS-domain was found to occur asymmetrically. In the case of the FLC–SVP heterodimer, the direction of asymmetrical DNA-binding in heterodimers was found to be fixed. Furthermore, the molecular dynamics simulations provided insight towards understanding the results generated from previous DPI-ELISA experiments, which should provide an improved means for predicting biologically significant CArG-boxes around genes.