Structure-function studies of the bacterial plasmid defence system Wadjet

微生物存在于许多环境中，几乎在所有情况下都受到噬菌体和其他自私遗传因子的持续攻击。为了避免感染，细菌进化出了一系列防御机制。这些不同的系统被发现聚集在基因组上，被称为“防御岛”。防御系统包括众所周知的限制性内切酶和CRISPR-Cas9，它们分别被用于分子生物学和基因编辑。然而，在防御岛上还有许多其他未被描述的系统，这些系统可能为科学家提供更多的工具来开发。该项目旨在利用生物物理学分析和结构生物学来探索以埃及女神Wadjet命名的一个这样的系统的机制。Wadjet系统防止细菌通过质粒转化。它包含四个基因：jetA，jetB，JetC和JetD。jetABC编码在序列水平上与细菌凝集素相关基因产物。凝聚素广泛存在于所有生物体中，是ATP依赖的分子机器，有助于调节细胞分裂过程中的基因组分离过程。它们的确切机制和作用仍有争议。它们形成大的蛋白质复合物，利用ATP水解来驱动DNA构象的大规模变化，包括将DNA拴在一起，或作为分子马达主动挤出DNA环。在Szczelkun和Berger-Schaffitzel实验室之间的这个合作项目中，您将确定Wadjet JetABCD复合物的整体结构，展示它如何与DNA相互作用，并确定ATP水解的作用。我们提出DNA捕获或环挤出阻断质粒复制。这些活动将直接区分和测量真实的时间使用单分子磁镊分析。

Microbes are found in many environments, and in almost all cases are under constant attack frombacteriophages and other selfish genetic elements. To avoid infection, bacteria have evolved a widerange of defence mechanisms. These different systems are found clustered on the genome in whatare called "defence islands". The defence systems include the well-know restriction enzymes andCRISPR-Cas9, which have been exploited for molecular biology and gene editing, respectively. Yetthere are many other uncharacterised systems within the defence islands that may provide yet moretools for scientists to exploit. This project aims to use biophysical analysis and structural biology toexplore the mechanism of one such system, named after the Egyptian goddess Wadjet.The Wadjet system prevents bacterial transformation by plasmids. It comprises four genes: jetA, jetB,JetC and JetD. jetABC encode gene products that are related at a sequence level to bacterialcondensins. Condensins are widespread across all organisms, and are ATP-dependent molecularmachines that help to regulate the process of genome segregation during cell division. Their exactmechanisms and roles are still debated. They form large protein complexes that use ATP hydrolysis todrive large-scale changes in DNA conformation, including tethering DNA together, or activelyextruding DNA loops as molecular motors. In this collaborative project between the Szczelkun andBerger-Schaffitzel labs, you will determine the overall structure of the Wadjet JetABCD complex, showhow it interacts with DNA, and determine the role for ATP hydrolysis. We propose that DNA captureor loop extrusion blocks plasmid replication. These activities will be directly distinguished andmeasured in real time using a single molecule magnetic tweezers assay.