Understanding the coordination of DNA mismatch repair using live-cell single-molecule imaging

从细菌到人类的所有生物都依赖分子机器来确保其基因组的精确复制。DNA错配修复途径（MMR）在DNA合成过程中发现并恢复错误。在细胞中执行MMR的蛋白质解决了一个显着的问题，即在基因组中数百万个正确匹配的碱基中检测单个错误结合的DNA碱基。没有MMR，细胞的突变率增加100到1000倍。因此，MMR缺失是疾病的驱动因素，基因变化加速导致人类癌症发展和病原体获得耐药性突变。因此，研究MMR的分子机制对于了解其在进化、健康和疾病中的基本作用至关重要。MMR还在基因组工程技术的效率方面发挥着重要作用，这些技术目前正在彻底改变生物研究、生物技术和医学。错配修复机制必须既快速又准确。这是通过不同蛋白质（称为MutS， MutL, MutH）的顺序作用来实现的，这些蛋白质的招募和DNA上的酶活性受到严格控制。基于纯化蛋白的实验，人们对MMR的机制已经有了很多了解，但修复过程如何在活细胞内起作用仍不确定。我们的建议解决了对MMR的理解上的这一巨大差距。我的实验室专门开发单分子显微镜方法，直接观察细胞中蛋白质的不受干扰的功能。通过高速荧光成像，我们可以追踪单个蛋白质的运动，并检测它们何时何地与DNA结合。这使我们能够获得解决MMR蛋白如何搜索修复位点以及如何协调途径中的顺序步骤所需的确切信息。我们将在大肠杆菌中进行这些实验，其中MMR已被最详细地表征。大肠杆菌细胞也非常适合我们的成像方法。可视化活细胞中的修复事件将使我们能够探索为什么MMR有时会出错。虽然这种情况很少发生，但每次修复失败都会导致细胞发生永久性突变。当人类细胞发生癌变或致病菌产生耐药性时，MMR通路就会出现故障。我们将从显微镜数据中提取有关修复事件的速度和位置的定量信息，并将其输入数学模型，以确定哪些因素决定了修复的成功和失败。总的来说，这个项目将确定MMR蛋白如何在一个途径中协同工作，为保存细胞遗传信息的中心过程提供直接的见解。

All organisms from bacteria to humans rely on molecular machines to ensure accurate replication of their genomes. The DNA mismatch repair pathway (MMR) spots and reverts errors during DNA synthesis. The proteins that perform MMR in cells solve a remarkable problem, detecting single misincorporated DNA bases amongst millions of correctly matched bases in the genome. Without MMR, mutation rates in cells increase 100 to 1000-fold. Loss of MMR is thus a driver of diseases, with accelerated genetic change leading to cancer development in humans and acquisition of drug resistance mutations in pathogens. Investigating the molecular mechanisms of MMR is therefore paramount for understanding its fundamental role in evolution, health and disease. MMR also plays an important role in the efficiency of genome engineering techniques that are currently revolutionising biological research, biotechnology, and medicine.The mismatch repair mechanism must be both fast and accurate. This is achieved via the sequential action of different proteins (called MutS, MutL, MutH), whose recruitment and enzymatic activities on DNA are tightly controlled. Much has been learnt about the mechanism of MMR based on experiments with purified proteins, but it remains uncertain how the repair process works inside a living cell. Our proposal addresses this large gap in the understanding of MMR. My lab specialises in the development of single-molecule microscopy methods to directly observe the unperturbed function of proteins in cells. Via high-speed fluorescence imaging, we can track the movement of individual proteins and detect when and where they bind to DNA. This allows us to obtain the exact information needed to address how MMR proteins search for repair sites and how the sequential steps in the pathway are coordinated. We will perform these experiments in E. coli bacteria - the organism in which MMR has been characterised in most detail. E. coli cells are also ideally suited for our imaging methods.Visualising repair events in living cells will allow us to explore why MMR sometimes goes wrong. Although this happens rarely, each repair failure leads to a permanent mutation in a cell. One failure of the MMR pathway can be the moment when a human cell turns cancerous, or a pathogenic bacterium becomes drug resistant. We will extract quantitative information about the speed and location of repair events from our microscopy data, and feed this into a mathematical model to identify which factors determine repair success and failure. Overall, this project will establish how MMR proteins work together in a pathway, providing direct insight into a central process that preserves the genetic information in cells.