Investigation of strategies and synergies in DNA lesion recognition using single molecule AFM imaging

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The stability of DNA is constantly chemically and physically threatened, resulting in structurally altered or chemically modified DNA that can lead to cancer, cell senescence or apoptosis. Different DNA modifications are repaired by a number of repair mechanisms that are fine-tuned to their particular target lesions. We use single molecule imaging by atomic force microscopy (AFM) to elucidate general and specific features of DNA damage recognition in different DNA repair systems. Here, we focus on alkyl-lesion repair by the alkyltransferase AGT and nucleotide excision repair (NER) induced by the alkyltransferase-like (ATL) protein. Initiation of NER by ATL proteins constitutes an interesting alternative of alkyl-lesion processing and a novel addition to the vast spectrum of NER targets. In addition to AFM, we exploit the synergistic approaches of analytical ultracentrifugation to address cooperative protein-DNA interactions in solution in particular with respect to differences in the AGT and ATL systems, and fluorescence optical tweezers to directly follow NER protein recruitment by the ATL system. We further address the generic question of how different properties of DNA lesions translate into initial lesion detection strategies of the important base excision repair DNA glycosylases. In support of our structural AFM analyses of DNA lesion substrates, we use fluorescence energy transfer (FRET) measurements. Glycosylases continuously probe the DNA for lesions in their target search, leading to an equilibrium between a search complex and a strongly bent interrogation complex. To access conformational properties of these different glycosylase-DNA complex states, we apply single molecule FRET (smFRET) using dSTORM. Together, AFM, ensemble FRET spectroscopy, and smFRET will provide powerful insight into mechanistic detail of lesion search and detection from structural and mechanical effects of glycosylase target lesions and their correlation with conformational properties of glycosylase-DNA complexes.