Untersuchung des DNA-Schneidemechanismus von Typ l Restriktionsenzymen

限制性内切酶是细菌抵御入侵病毒的主要防御机制。它们根据目标序列的甲基化状态识别病毒DNA，并将其切割成碎片。I型限制性内切酶由一个甲基转移酶核心单元组成，负责DNA结合，它可以结合多达两个运动亚基。在病毒DNA靶点上，这些运动亚基开始以atp驱动的方式结合和转移邻近的DNA，从而形成大的DNA环。切割是随机发生的，远离目标位点，被认为是在与第二个易位酶碰撞时发生的。虽然II型内切酶的切割机制已经被很好地理解，但迄今为止，对于I型内切酶如何切割DNA知之甚少。因此，在这个项目中，我们想要研究这些酶的裂解机制。利用最先进的单分子技术，特别是磁镊子结合荧光显微镜，我们想直接观察单个酶复合物在易位和裂解过程中，直接测试和探索碰撞模型。

Restriction enzymes are the main defence mechanism of bacteria against invading viruses. They recognize viral DNA upon the methylation state of their target sequence and cleaving it into pieces. Type I restriction enzymes consist of a methyltransferase core unit, responsible for DNA binding, which can bind up to two motor subunits. On viral DNA target sites, these motor subunits start to bind and translocate adjacent DNA in an ATP-driven manner, which leads to the formation of large DNA loops. Cleavage occurs randomly, distant from the target sites and is thought to occur upon collision with a second translocating enzyme. While the cleavage mechanism of Type II restriction enzymes is quite well understood, little is so far known on how Type I restriction enzymes cleave DNA. Within this project we therefore want to investigate the cleavage mechanism of these enzymes. Using state-of-the-art single-molecule techniques, in particular magnetic tweezers combined with fluorescence microscopy, we want to directly observe individual enzymes complexes during translocation and cleavage to directly test and explore the collision model.