Biophysical studies of DNA binding – by the large filament-forming protein Rad51 and the small minor-groove binder Hoechst 33258

Doktorsavhandling, 2015

Mechanistic insight into the nature of DNA-binding ligands is crucial for both drug development as well as understanding more complex biological reactions that take place in the cell. In this Thesis, two rather different DNA-binding molecules are considered: 1. the large, filament-forming eukaryotic recombination protein Rad51, which is essential in the strand exchange reaction during homologous recombination, the most accurate repair system of DNA double‑strand breaks, and 2. the small, synthetic DNA ligand Hoechst 33258, which is a model drug for DNA minor groove interactions. The Rad51 filament formation, reflected in the length of short individual Rad51 filament patches on long DNA strands, has been examined by nanofluidics in combination with fluorescence microscopy. Analyses of the dynamics of the Rad51-DNA complex in the nanochannel reveal structural variations that depend on the filament formation conditions; the choice of divalent cations (Mg2+ or Ca2+), the DNA substrate (single- or double‑stranded), and the Rad51 nucleation concentration affected the macroscopic structure of the filament. The structural effects that the divalent cations Mg2+ and Ca2+, and the accessory protein Swi5-Sfr1 exert on the Rad51-single-stranded (ss) DNA filament at a microscopic level have also been examined by linear dichroism (LD). The naturally unordered bases in ssDNA become preferentially perpendicularly oriented relative to the DNA backbone in presence of Rad51 with Ca2+ alone, or Mg2+ in combination with the accessory protein Swi5-Sfr1. A preferentially perpendicular base organization is proposed to mechanistically relate to an efficient strand exchange reaction, supposedly due to more critical base matching with the invading double‑stranded DNA. To aid future spectroscopic structural analyses of proteins that contain tyrosine residues, such as Rad51, a combined spectroscopic and in silico study of the chromophore in tyrosine has been conducted. It is demonstrated how the spectroscopic properties of tyrosine are sensitively dependent on the polarity of the environment, mainly through the ability to form hydrogen bonds and the rotation of the hydroxyl group. The last part of the Thesis deals with spectroscopic and thermodynamic studies of the binding of Hoechst 33258 to three different DNA oligonucleotides with AT‑tracts of various lengths. The binding at high drug‑to‑DNA ratio is especially considered, and an important conclusion is that two Hoechst 33258 molecules bind in parallel, a slight distance apart, in the minor groove of an oligonucleotide with 8 consecutive adenines/thymines.