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

细胞每次分裂时，都必须复制其遗传物质，这样每个子细胞才能获得一套完整的基因。在复制过程中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产生致命的后果。不幸的是，目前存在许多障碍，可能会阻碍这一复制过程。我们已经发现，一个主要问题是包裹DNA的许多蛋白质，这些蛋白质是包装、阅读和修复遗传物质的正常过程所需的。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。这种阻碍也可能触发突变，因为当复制机器停止时，遗传物质内的重排会被诱导。然而，我们最近的工作表明，辅助马达有助于清除前进中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，这些辅助马达与复制机器的物理相互作用对于它们的正常功能至关重要。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，以及附属马达如何改变这一运动，以确定这些机器在遇到蛋白质障碍时如何反应。研究单个复制复合体是至关重要的，因为不同DNA分子的复制是不同步的。同时测量大量分子不会显示电机速度的差异，也不会显示单个电机的停顿，而只会显示平均重复速率。此外，我们知道，当比较单个复合体时，可以看到行为上的巨大差异。对于完成准确的基因组复制来说，罕见的行为可能是非常重要的。通过观察单个复合体，罕见事件更容易被发现。我们将使用一种成像技术，当单个复制复合体复制DNA时，可以检测到它们，从而允许监测单个复合体对蛋白质屏障的反应。我们还将研究辅助马达与复制机械的物理相互作用如何帮助辅助马达发挥作用。我们假设，这种相互作用刺激了酶不同部分之间的相对运动，激活了运动功能，并帮助在复制机器之前清除蛋白质。我们将使用一种技术，非常准确地测量单个分子内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导致辅助马达内的运动，以及这种相互作用是否刺激运动活动。DNA的复制从细菌到人类是高度保守的，蛋白质屏障是所有有机体的问题。我们的工作将确定这一普遍问题如何影响DNA复制过程，以及细胞如何减少蛋白质屏障对DNA复制的影响。了解细胞是如何克服DNA复制障碍的，也将有助于设计针对有效复制所需的辅酶的药物。这类药物可能会作为治疗传染病的新抗生素和治疗癌症的化疗药物等潜在应用。新的合成生物体的设计，例如帮助生物燃料生产，也将从这个项目中受益。新的生物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复制，才能使有机体生长和分裂。了解细胞如何在蛋白质障碍面前复制其遗传物质，将有助于设计这种新型有机体。

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.