DNA-Complexes with Drugs and Proteins

Licentiate thesis, 2012

DNA is fundamental for all living cells; the DNA holds the genetic code, which is more or less the instruction book for how all cells are built and function. Several diseases are also linked to DNA, emerging either from a mutation in our genome, which could result in a malfunctioning protein, or that the transcription of genes is somehow affected by structural changes in the DNA, induced by mutations or DNA binding molecules. Research concerning how both small molecules and larger protein assemblies bind to the DNA are therefore of great interest since these could be used as future drugs in for example gene therapy. In the first part of this Thesis the non-covalent binding to DNA of a small minor groove binder, Hoechst 33258, is examined. The molecule is rather well-studied, but there are still questions concerning its multiple binding modes to DNA sequences rich in adenines (A) and thymines (T) that remain unanswered. An increased understanding of the nature of the multiple binding modes could benefit the future design and development of sequence specific drugs. Using the thermodynamic characterization of the binding through Isothermal Titration Calorimetry (ITC) in combination with the spectroscopic properties of the formed complexes through Circular Dichroism (CD) we have analyzed the experimental results in a global dataset. We conclude that two molecules of Hoechst 33258 can bind next to each other in AT-rich sequences that consist of eight AT base pairs, but not in sequences consisting of six or less AT base pairs. They do not bind on top of one another, in the form of a sandwich, as previously proposed, nor contiguously, but with distinct separation between monomeric units. The second part of this Thesis reports how the structure and activity of the human recombination protein RAD51 (HsRad51) depends on presence of cofactors: ATP and divalent cations. The eukaryotic HsRad51 is one of the evolutionarily best-conserved proteins and homologues to it can be found in both Bacteria and Archaea. HsRad51 is involved in the strand exchange reaction of homologous recombination, which takes place during meiosis and repair of double-strand breaks in eukaryotes. With further understanding of the strand exchange reaction we might find ways to utilize it in the medicinal field, such as for correction and repair of defective genes in gene therapy, or as a potential target in cancer treatment. We confirm that the first intermediate of this reaction, in which HsRad51 forms a helical filament around a single strand of DNA, demonstrates a perpendicular organization of the DNA bases relative the filament axis when ATP and Ca2+ are present. This organization is most probably related to the observed high strand exchange activity of the HsRad51/ssDNA complex in with ATP and Ca2+. By contrast, in presence of Mg2+ we observe both poor base organization and strand exchange activity.