Obstacles to replication: uncovering the mechanisms of macromolecular collisions

DNA包含生命所需的信息。所有的有机体在生长或繁殖之前都必须复制它们的DNA。遗传信息是使用一种名为复制体的分子机器复制的，它在沿着DNA旅行时复制信息。为了防止DNA中的信息损坏，这种复制过程必须准确发生。当复制体沿着DNA移动时，它可能会被几种类型的障碍所阻止。最常见也是最有问题的例子是另一台名为RNA聚合酶的分子机器。RNA聚合酶读取DNA中编码的基因，以帮助制造细胞所需的分子。在细菌中，复制体和RNA聚合酶同时活跃，这可能导致DNA发生碰撞。这些分子机器的碰撞会破坏DNA，进而导致细胞死亡。此外，还有其他障碍也可以阻止复制体。终止是DNA复制的最后一步，当向相反方向移动的DNA复制机制相遇时发生。在大肠杆菌中，Tus蛋白与DNA结合，形成单向复制屏障。复制体可以保持在这个屏障上，直到终止发生。在这个项目中，我们将研究当复制体与这两个区块碰撞时会发生什么。复制体与障碍的碰撞可以最小化，但不能避免。细胞含有几种名为解旋酶的酶，可以解开DNA并解决碰撞。有几种解旋酶已经被鉴定出来，但我们不知道它们是如何工作的。我们的目标之一是了解为什么解旋酶可以解决一些阻塞，而不是其他阻塞，以及为什么它们的效率不同。为了做到这一点，我们将使用TUS或RNA聚合酶作为复制体阻滞工具，然后使用先进的显微镜来可视化被阻止的蛋白质。识别在这些障碍下受阻的复制机制的特征将有助于我们理解碰撞时发生的事情以及它们是如何解决的。我们还将确定复制机器在与障碍物相撞后保持组装的时间，并测量机器与RNA聚合酶区块的距离。复制障碍是所有生命形式的一个问题，但我们将在大肠杆菌中研究这些过程，因为它们很容易操作，而且它代表了一个很好的模型系统。来自大肠杆菌的见解通常与包括人类在内的所有生命相关，因为我们的许多蛋白质是相似的。我们的结果将帮助我们理解当存在障碍时DNA是如何复制的。了解细菌对这个问题的解决方案将使我们深入了解人类的同样过程。从长远来看，这项工作可能会对人类健康研究产生影响，因为相关的解旋酶与癌症易感性、人类疾病和衰老有关。抗菌药耐药性的演变是医学上的一个巨大问题，因为抗生素被用来治疗许多疾病，并使手术变得安全。我们将研究的解旋酶在细菌中很常见，对它们的生存至关重要。了解它们的工作原理有助于使它们成为抗微生物药物的新靶点。针对DNA复制等重要过程的抗生素治疗可以改善患者的预后，减少耐药性演变的机会。此外，研究表明，当化疗药物与靶向解旋酶的药物一起使用时，效果会更好。因此，从这个项目中获得的知识可能会在未来惠及多个患者群体。我们计划使用的先进显微技术在一个迅速发展的领域是一项令人兴奋的技术。一个直接的好处是，该项目将通过培训科学家掌握专业技术和数据分析，帮助减少英国在这一领域的技能缺口。

DNA contains information required for life. All organisms must copy their DNA before they can grow or reproduce. Genetic information is copied using a molecular machine, called the replisome, which copies information while travelling along DNA. To prevent corruption of the information in DNA, this duplication process must occur accurately. When the replisome is travelling along DNA it can be blocked by several types of obstacles. The most common and problematic example is another molecular machine called RNA polymerase. RNA polymerase reads the genes encoded in DNA to help produce molecules required by the cell. In bacteria the replisome and RNA polymerase are both active at the same time, which can lead to collisions on the DNA. Collisions of these molecular machines can damage DNA and this in turn can cause cell death. Furthermore there are other barriers that can also block the replisome. Termination is the final step of DNA replication and occurs when DNA replication machinery travelling in opposite directions meet. In the bacterium Escherichia coli, the Tus protein binds DNA and forms a one-way replication barrier. The replisome can be held at this barrier until termination occurs. In this project we will investigate what happens when the replisome collides with both of these blocks.Replisome-obstacle collisions can be minimised but not avoided. Cells contain several enzymes called helicases which can unwind DNA and resolve the collisions. Several helicases that do this job have been identified but we do not know exactly how they work. One of our goals is to understand why helicases can resolve some blockages but not others and why their efficiency varies. To do this we will use Tus or RNA polymerase as a replisome stalling tool and then use advanced microscopy to visualise the blocked proteins. Identifying the features of replication machinery blocked at these obstacles will help us to understand what happens at collisions and how they are resolved. We will also determine how long the replication machinery remains assembled once it has collided with an obstacle and measure how close the machinery can get to a RNA polymerase block. Replication obstacles are a problem for all life forms, but we will study these processes in E. coli bacteria since they are easy to manipulate and it represents an excellent model system. Insights from E. coli often have relevance to all life, including humans, because many of our proteins are similar.Our results will help us understand how DNA is replicated when there are obstacles in the way. Understanding bacterial solutions to this problem will give us insight into the same process in humans. In the long term this work can have an impact on human health research because related helicases have been implicated in cancer predisposition, human disease and ageing. The evolution of resistance to anti-microbial drugs is a huge problem in medicine because antibiotics are used to treat many diseases and also enable safe surgery. The helicases we will investigate are common amongst bacteria and can be essential for their survival. Understanding how they work could help make them new targets for anti-microbial drugs. Antibiotic treatment targeting important processes like DNA replication could improve patient outcomes and reduce the chance of resistance evolving. In addition, it has been shown that chemotherapeutic agents are more efficient when administered alongside drugs that target helicases. The knowledge gained from this project could therefore benefit multiple patient groups in the future. The advanced microscopy we plan to use is an exciting technique in a rapidly expanding field. An immediate benefit is that this project will help reduce the UK skills deficit in this area by training scientists in the specialised techniques and data analysis.