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

DNA的稳定性不断受到化学和物理威胁，导致结构改变或化学修饰的DNA，可导致癌症，细胞衰老或凋亡。不同的DNA修饰通过许多修复机制进行修复，这些修复机制针对其特定的靶病变进行微调。我们使用原子力显微镜（AFM）的单分子成像来阐明不同DNA修复系统中DNA损伤识别的一般和具体特征。在这里，我们专注于烷基损伤修复的烷基转移酶AGT和核苷酸切除修复（NER）诱导的烷基转移酶样（ATL）蛋白。由ATL蛋白启动NER构成了烷基损伤处理的一个有趣的替代方案，并且是对广泛的NER靶点的一个新的补充。除了原子力显微镜，我们利用分析ultracentrugation的协同方法，以解决合作的蛋白质-DNA相互作用在溶液中，特别是相对于AGT和ATL系统的差异，和荧光光镊直接遵循NER蛋白质招募ATL系统。我们进一步解决的一般性问题，如何不同的性质的DNA病变转化为初始病变检测策略的重要碱基切除修复DNA糖基化酶。为了支持我们的DNA损伤基板的结构AFM分析，我们使用荧光能量转移（FRET）测量。糖基化酶在它们的目标搜索中连续地探测DNA的损伤，导致搜索复合物和强烈弯曲的询问复合物之间的平衡。为了获得这些不同的糖基化酶-DNA复合物状态的构象特性，我们使用dSTORM应用单分子FRET（smFRET）。总之，原子力显微镜，合奏FRET光谱，和smFRET将提供强大的洞察损伤搜索和检测的结构和机械影响的糖基化酶靶病变及其相关性与糖基化酶-DNA复合物的构象特性的机制细节。

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.