Untersuchung des DNA-Schneidemechanismus von Typ l Restriktionsenzymen

限制性内切酶是细菌抵御病毒入侵的主要防御机制。他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负责DNA结合的甲基转移酶核心单位组成，它可以结合多达两个马达亚基。在病毒DNA靶点上，这些运动亚基开始以ATP驱动的方式结合和移位相邻的DNA，从而导致大的DNA环的形成。切割是随机发生的，距离目标位置很远，被认为是在与第二个转运酶碰撞时发生的。虽然人们对II型限制性内切酶的切割机制相当了解，但到目前为止，人们对I型限制性内切酶如何切割DNA知之甚少。因此，在这个项目中，我们想要研究这些酶的切割机制。利用最先进的单分子技术，特别是磁钳与荧光显微镜相结合，我们希望直接观察移位和切割过程中的单个酶复合体，以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.