Speeding and stuttering: analysing the dynamics of DNA replication at the single molecule level

每当细胞分裂时，它必须复制其遗传物质，以便每个子细胞获得一套完整的基因。在复制过程中所犯的任何错误都可能是灾难性的，因为即使是一个错误也可能导致致命的后果。不幸的是，目前有许多障碍可以阻止这一复制过程。我们发现一个主要的问题是包裹在DNA表面的许多蛋白质，这些蛋白质是包装、读取和修复遗传物质的正常过程所需要的。当这些蛋白质与DNA模板结合时，可以阻止DNA的复制并阻止复制过程的完成。这种阻断也可能引发突变，因为当复制机器停止时，遗传物质内部的重排会被诱导。然而，我们最近的工作表明，辅助马达有助于清除前进的复制机器路径上的蛋白质，在正常的DNA复制中起着至关重要的作用，并且这些辅助马达与复制机器的物理相互作用对其正常功能至关重要。了解复制机器如何沿着蛋白质包裹的DNA移动是很重要的。这些运动模式将决定完成基因组复制的可能性和发生突变的可能性。在这个项目中，我们将发现单个复制机器如何沿着蛋白质包裹的DNA移动，以及辅助马达如何改变这种运动，以确定这些机器在遇到蛋白质屏障时如何反应。研究个体复制复合体是必要的，因为不同DNA分子的复制是不同步的。同时测量大量的分子不会显示出马达速度或单个马达暂停的差异，而只能显示出平均的复制率。此外，我们知道，在比较个体复合体时，可以看到行为上的巨大差异。罕见的行为可能对完成精确的基因组复制非常重要。罕见事件更容易通过观察个别复合体来发现。我们将使用一种成像技术，可以检测复制DNA的单个复制复合体，从而监测单个复合体对蛋白质屏障的反应。我们还将研究辅助电机与复制机械的物理相互作用如何帮助辅助电机发挥作用。我们假设这种相互作用刺激酶不同部分之间的相对运动，激活运动功能，帮助在复制机器之前清除蛋白质。我们将使用一种技术，非常精确地测量单个分子中两个不同位置之间的距离，以确定与复制机制的相互作用是否会诱导副马达内的运动，以及这种相互作用是否会刺激运动活动。从细菌到人类的DNA复制是高度保守的，蛋白质屏障对所有生物都是一个问题。我们的工作将确定这个普遍问题是如何影响DNA复制过程的，以及细胞如何减少蛋白质屏障对DNA复制的影响。了解细胞如何克服DNA复制的障碍也将有助于设计针对有效复制所需的辅助酶的药物。这类药物有可能作为治疗传染病的新型抗生素和治疗癌症的化疗药物。新的合成生物的设计，例如帮助生物燃料生产，也将从这个项目中受益。新的有机体必须包含维持细胞生命的遗传指令，这些遗传指令必须能够被准确地复制，以使有机体生长和分裂。了解细胞如何在面对蛋白质屏障时复制其遗传物质将有助于设计这种新型生物。

Every time a cell divides it must copy its genetic material so that each daughter cell receives a complete set of genes. Any mistakes made during this copying process can be disastrous as even a single mistake can have fatal consequences. Unfortunately many obstacles are present that can block this replication process. We have discovered that a major problem are the many proteins that coat the DNA and that are needed for the normal processes of packaging, reading and repairing the genetic material. These proteins, when bound to the DNA template, can block replication of the DNA and prevent completion of the copying process. Such blockage may also trigger mutations since rearrangements within the genetic material are induced when replication machines come to a halt. However, our recent work has shown that accessory motors help to clear proteins out of the path of the advancing replication machine, playing a vital role in normal DNA replication, and that physical interaction of these accessory motors with the replication machinery is critical for their normal function.Understanding how replication machines move along protein-coated DNA is important. These patterns of movement will dictate the likelihood of completing genome duplication and the probability of mutations occurring. In this project we will discover how individual replication machines move along protein-coated DNA, and how accessory motors alter this movement, to establish how these machines react upon encountering protein barriers. Studying individual replication complexes is essential because the replication of different DNA molecules is not synchronised. Measuring a large number of molecules at the same time will not reveal differences in motor speed nor pausing of individual motors, but only an average rate of duplication. In addition, we know that wide variations in behaviour are seen when comparing individual complexes. Behaviours which are rare might be very important with respect to completion of accurate genome duplication. Rare events are much easier to detect by observing individual complexes. We will use an imaging technique that can detect single replication complexes as they duplicate DNA, allowing the responses of individual complexes to protein barriers to be monitored. We will also investigate how physical interaction of an accessory motor with the replication machinery helps the accessory motor to function. We hypothesise that this interaction stimulates relative movement between different parts of the enzyme, activating the motor function and helping to clear proteins ahead of the replication machine. We will use a technique that measures the distance between two different positions within a single molecule very accurately to determine whether interaction with the replication machinery induces movements within the accessory motor and whether this interaction stimulates motor activity.Copying of DNA is highly conserved from bacteria to man and protein barriers are a problem for all organisms. Our work will identify how this universal problem affects the DNA copying process and the means by which cells reduce the impact of protein barriers on DNA copying. Understanding how cells overcome barriers to DNA copying will also help in the design of drugs that target the accessory enzymes needed for efficient copying. Such drugs could have potential applications as new antibiotics for the treatment of infectious diseases and chemotherapy agents for the treatment of cancer. The design of new synthetic organisms, for example to aid biofuel production, will also benefit from this project. New organisms must contain the genetic instructions to maintain the life of that cell and these genetic instructions must be able to be copied accurately to allow the organism to grow and divide. Understanding how cells copy their genetic material in the face of protein barriers will help with the design of such novel organisms.