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Intact and Damaged DNA and their Interaction with DNA-Binding Proteins: a Single Molecule Approach
(2004)
- In the present work we study the architecture of intact and damaged DNA and their interaction with DNA-binding proteins using a combination of atomic force microscopy (AFM) and fluorescence correlation spectroscopy (FCS). In the beginning of this scientific work we therefore focus our efforts on the development of a reproducible protocol for the surface-deposition of different biomolecules, such as linear and circular DNA, different types of proteins and DNA-protein complexes. The analysis of the apparent contour length of intact DNA molecules of different lengths and under different preparation routines is found to be consistent with its B-DNA conformation. In this thesis we report about the first structural study of long DNA molecules that carry UV-light photolesions at random sites. An exposure of DNA to UV light introduces different modifications to the DNA structure: regions of unpaired DNA of different lengths, sharp kinks, the presence of knot like structures and single-strand breaks. In addition, the dynamics of damage accumulation can be traced with AFM. UV-exposure influences both the apparent contour length and the persistence length of the molecules. The structure of UV light damaged DNA strongly depends on the exposure time. Longer UV-light exposure time introduces increasing DNA damage. An FCS study on DNA exposed to UV-light shows that the presence of photoadducts influences the hydrodynamic properties of DNA molecules. The results obtained using both AFM and FCS are compared with gel-electrophoretical experiments. Further, we report about a first AFM study of the hRPA binding properties to intact and damaged DNA. The damage types under investigation were a 6 nt bubble modification, a cisplatin modification as well as lesions induced by UV-light. In our AFM experiment we never find RPA to be bound to the intact dsDNA chain. In complexes with intact DNA human RPA is only found to be bound to the termini of the linear DNA molecules. Human RPA binds with a very low affinity to both DNA containing a 6 nt bubble modification and a single cisplatin modification. Moreover, hRPA showed in this case still a preferential binding to the ends of the linear DNA molecules as seen in the complexes with intact DNA. Because of the low binding strength of hRPA to the cisplatin intrastrand adduct it is easily possible to remove the protein from its DNA complexes by scanning movements of the AFM tip. The images of the DNA molecules after removal of the hRPA proteins show significant distortions of the DNA chain, namely: the separation of the double strand into single-strands resulting in a large region of unpaired bases. Such structures are never observed in images of DNA carrying a cisplatin lesion before the addition of hRPA. This fact may explain a possible role of hRPA in damage excision. Our experiments show a rather high affinity of hRPA to UV-light damaged DNA that increases with UV-light exposition time. The formation of complexes of hRPA and UV-light damaged DNA molecules was studied by both EMSA and AFM. When hRPA was added to the damaged DNA, globular objects sitting on the rod shaped DNA strands were regularly observed in the AFM images. A systematic analysis of the apparent contour length of DNA molecules in protein-DNA complexes reveals a reduction of the contour length in comparison to the one of uncomplexed DNA molecules damaged for the same time. Such reduction of 27.7±7.6 nm suggests a wrapping of the DNA molecule around the hRPA protein. Additionally, using the hRPA-DNA system, we show that the application of the phase signal allows to differentiate between components of similar architecture, but different origin within one AFM image. The last chapter of the thesis is dedicated to the analysis of the DNA-binding properties of ORF80, a novel leuzine protein of unknown physiological role. Our AFM measurements demonstrate that under low protein concentration a specific binding of ORF80 dominates. To the best of our knowledge ORF80 is the smallest protein that was resolved in complexes with DNA by AFM. The single molecule approach in the study of the ORF80 binding properties to DNA with AFM shows that one, at most two ORF80 monomers recognize a specific sequence on the dsDNA. Both AFM and FCS clearly show that higher ORF80 concentrations lead to the formation of big protein-DNA agglomerates that contain numerous DNA and protein molecules. The formation of these agglomerates can be explained both by the unspecific ORF80 binding and its high aggregation properties.
