How bacteria replicate their DNA in spite of barriers, one molecule at a time

如果制造和维持一个生物体所必需的信息包含在一本书中，那么DNA就代表了这本书的字母——实际上，它就是生命的字母表。任何生物体的一个关键特征就是复制这些字母的能力，要么复制更多的自己，要么通过创造子细胞来创造后代。这个复制DNA的关键过程就是“DNA复制”。它涉及到分子纳米机器，这些机器沿着DNA运行，就像汽车沿着道路行驶一样，迫使DNA的双螺旋打开，然后复制分开的链。然而，这些纳米机器的有效运行存在许多分子障碍，可能导致碰撞，如果细胞不纠正可能是致命的。但是这种复制纳米机器在碰撞时会发生什么呢？它们是脱落、分裂，还是帮助它们穿过屏障，或者是在屏障的另一边建造了新的纳米机器，然后可以不受阻碍地移动？细胞已经进化出了一套非凡的策略，可以修复这些纳米级的碰撞，从而使DNA复制仍然可以进行。其他科学家此前在帮助我们理解DNA修复过程是如何实现方面做了大量工作，但他们中的大多数人研究的是许多细胞的群体，他们研究的是许多分子的平均值，而不是单个细胞中的单个纳米机器，主要是因为观察细胞中的分子的技术还不够好——直到现在！在这里，我们将使用细菌模式生物大肠杆菌，我们可以可视化和跟踪单个纳米机器。我们还将在试管中纯化DNA和复制纳米机器以及屏障，然后在显微镜下观察单分子。这两种互补的方法将使我们能够将纯化组分的分子尺度过程的观察结果以及在真实复杂的细胞环境中实际发生的情况拼凑在一起。这将使我们能够详细地看到复制纳米机器碰撞时的细节，以及其他分子如何做出反应来修复碰撞。这种革命性的方法将与新的、令人兴奋的实时“碰撞电影”分析相结合，与人工智能或“AI”相结合——这种软件由复杂的交互代码层组成，类似于大脑中神经细胞在视觉皮层中连接在一起的方式。每一个这样的神经对输入进行特定的数学运算，并将输出传递给下一层的神经细胞。通过这样做，神经细胞网络可以被“训练”来识别图像中的关键特征和模式，这对于我们在试管和活细胞中的单分子显微镜中相对嘈杂的图像数据非常有用，可以告诉我们不同的分子在哪里以及它们如何相互作用。我们的工作将告诉我们，如果复制纳米机器被障碍物阻挡，甚至被推开，是什么帮助它们回到DNA的道路上。此外，非常重要的是，它将使我们了解靶向DNA复制和修复的抗生素是如何在细胞中起作用的。这将是重要的信息，因为许多所谓的“超级细菌”正在出现，它们对抗生素不再有反应，因此这可能有助于其他研究人员能够设计出新型更好的抗生素。跟踪单分子并建立更好的技术，正如我们在这里所做的那样，将使任何参与任何细胞或生物体DNA复制的科学家基本上都能改进他们的工作。

If the information necessary to make and maintain a living organism were contained inside a book, then DNA would represent the book's letters - it is, in effect, the alphabet of life. A key feature of any organism is the ability to replicate these letters, either to make more of itself or to create progeny by creating daughter cells. This crucial process of copying DNA is "DNA replication". It involves molecular nanomachines, which run along DNA like a car runs along a road, forcing open its double-helix, then making copies of the separate strands. However, many molecular barriers exist to the efficient running of these nanomachines potentially causing collisions that if the cell did not correct could be lethal. But what happens with such a replication nanomachine at collisions? Do they drop off, fall apart, are they helped to push through the barrier, or are new nanomachines built the other side of the barrier that can then move on unimpeded?Cells have evolved a suite of remarkable strategies that allow these nanoscale collisions to be repaired so that DNA replication can still occur. Other scientists have done great work previously in helping us to understand how DNA repair processes are achieved, but most of them have studied populations of many cells looking at an average of many molecules, instead of just individual nanomachines in single cells, mainly because the technology available to look at molecules in cells has not been good enough - until now! Here we will use the bacterial model organism Escherichia coli in which we can visualise and track individual nanomachines. We will also purify DNA and the replication nanomachines and barriers in the test tube and look at these at a single-molecule in the microscope. These two complementary approaches will really allow us to piece together observations of the molecular scale processes of just the purified components as well as what actually happens inside real, complex cell environments. This will allow us to see in exquisite detail when replication nanomachines collide, and how other molecules then respond to repair the collision. This revolutionary approach will be combined with new, exciting analysis of our real time "collision movies", with Artificial Intelligence or "AI" - this software consists of complex layers of interacting code, similar to the ways that nerve cells in the brain link together in the visual cortex. Each such nerve performs specific maths operations on inputs and passes the outputs to nerve cells in the next layer. In doing so a network of nerve cells can be "trained" to recognise key features and patterns from images, which can be really useful for the relatively noisy image data that we have in single-molecule microscopy both in test tubes and in living cells, to tell us where different molecules are and how they interact with each other. Our work will tell us what helps the replication nanomachines back on the DNA road if they have been blocked by an obstacle or even pushed off. Also, very importantly, it will allow us to understand how antibiotics which target DNA replication and repair actually work in cells. This will be important information, since many so-called "super-bugs" are emerging which no longer respond to antibiotics, and so this may aid other researchers in being able to design new types of better antibiotics. Following single molecules and establishing better techniques, as we aim to do here, will enable basically any scientist involved in DNA replication in any cells or organism to improve their work.