Structure-function studies of the bacterial plasmid defence system Wadjet

微生物存在于许多环境中，几乎所有情况下都不断受到噬菌体和其他自私遗传因素的攻击。为了避免感染，细菌进化出了更广泛的防御机制。这些不同的系统被发现聚集在基因组上被称为“防御岛”的地方。防御系统包括广为人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，在防御岛屿内还有许多其他不寻常的系统，可能会为科学家提供更多的工具来开发。该项目旨在使用生物物理分析和结构生物学来探索这样一个系统的机制，该系统以埃及女神瓦杰特的名字命名。瓦杰特系统防止通过质粒进行细菌转化。它由四个基因组成：Jeta、JetB、JetC和JetD。JetABC编码在序列水平上与细菌凝集素相关的基因产物。凝聚素广泛存在于所有生物体中，是一种依赖于ATP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存在争议。它们形成大型蛋白质复合体，利用三磷酸腺苷的水解来驱动DNA构象的大规模变化，包括将DNA捆绑在一起，或主动挤出DNA环作为分子马达。在Szczelkun和Berger-Schaffitzel实验室之间的这个合作项目中，您将确定WadJet JetABCD复合体的整体结构，了解它如何与DNA相互作用，并确定ATP水解的作用。我们认为DNA捕获或环挤出阻止了质粒复制。这些活动将被直接区分并使用单分子磁镊法进行实时测量。

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.