Final Report Summary - DYNASING (Dynamic single-molecule approach to DNA homologous recombination) IntroductionHomologous recombination, the exchange of sequences between homologous DNA molecules, is essential for accurate genome duplication, DNA damage repair and chromosome segregation. Single molecule analysis provides information on intermediate states, functional and structural variability and the distribution of variable states that cannot be recovered from bulk biochemical assays.Recombination proteins labelled with a flourophore has been used in single molecule fluorescence microscopy assays and scanning force microscopy (SFM). The main objectives are:1-analyse the dynamic rearrangements between DNA and Rad51 to gain insight into the key events that drive DNA strand exchange;2-analyse the effect of accessory factors of Rad51 on its assembly/disassembly from DNA to gain insight into mechanisms that limit homologous recombination to appropriate locations.MethodsnSFM is an ideal tool for direct imaging, providing 3D information on molecular structure at nm resolution from defined components. The images reveal structural details on the size and location of DNA bound proteins as well as protein-induced arrangement of the DNA, which are directly correlated in the same complexes. Combining scanning force and fluorescent microscopy allows simultaneous identification of labelled biomolecules and analysis of their nm level architectural arrangement. Fluorescent polystyrene nano-spheres were used as reliable objects for alignment of optical and topographic images. This allowed the precise localisation of different fluorescence particles within complex molecular assemblies whose structure was mapped in nanometre detail topography.In a different approach, we analysed the movement of purified human RAD54 protein, an accessory factors of Rad51, with nanometre and millisecond resolution by using fast-SFM. In collaboration with Kyoto University, we made use of this state of the art technology for studying single molecule dynamics with unprecedented spatial and temporal resolution.ResultsWe have described new methods and procedures for simple and reliable imaging of biomolecules. This allowed the precise localisation of multicolour fluorescence particles within complex molecular assemblies whose structure was mapped in nanometre detail topography. This is achieved by simultaneous SFM and fluorescence microscopy, which together produce nm resolution structural information and use fluorescent tags to identify different molecular components. This combination of techniques will allow a wealth of new applications particularly in understanding the architecture of complex DNA-protein assemblies. We already have applied the technique for studying the interaction of BRC2 sequence with RAD51-DNA filaments (in preparation).We have showed the successful expression and purification of a stable complex containing only RAD50 and NBS1 (RN). The RAD50/MRE11/NBS1 protein complex (RMN) plays an essential role during the early steps of DNA double-strand break (DSB) repair by homologous recombination. Although MRE11 is a DNA-binding protein it was not required for DNA binding per se or DNA-tethering activity of the complex.We have analysed the movement of purified human RAD54 protein, an accessory factor of Rad51, with nanometre and millisecond resolution by using fast-SFM. We traced the dsDNA-dependent ATPase action of the protein after releasing caged ATP with UV light.
