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

细菌如何克服障碍，一次复制一个分子的 DNA

基本信息

批准号：
BB/W000555/1
负责人：
Mark Leake
金额：
$ 54.54万
依托单位：
University of York
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2021
资助国家：
英国
起止时间：
2021 至无数据
项目状态：
未结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FW000555%2F1
关键词：
How bacteria replicate their DNA

项目摘要

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.

如果一本书中包含了制造和维持一个生物体所必需的信息，那么DNA将代表这本书的字母--它实际上是生命的字母表。任何生物体的一个关键特征是复制这些字母的能力，要么是为了制造更多的自己，要么是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，就像汽车沿着道路行驶一样，沿着DNA行驶，迫使它的双螺旋打开，然后复制单独的链。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，如果细胞不正确，可能是致命的。但是这样一台复制纳米机器在碰撞时会发生什么呢？它们会脱落、解体吗？它们会被帮助穿过屏障吗？还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继续前进？细胞已经进化出一套非凡的策略，可以修复这些纳米级的碰撞，使DNA复制仍然可以发生。其他科学家以前在帮助我们了解DNA修复过程是如何实现的方面做了大量工作，但他们中的大多数人都研究了许多细胞的群体，而不仅仅是单个细胞中的单个纳米机器，主要是因为可用于观察细胞中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和复制纳米机器和屏障，并在显微镜下观察单个分子。这两种互补的方法将真正使我们能够将对纯化成分的分子尺度过程的观察以及真实的复杂细胞环境中实际发生的事情拼凑在一起。这将使我们能够看到复制纳米机器碰撞时的精致细节，以及其他分子如何响应以修复碰撞。这种革命性的方法将结合新的，令人兴奋的分析我们的真实的时间“碰撞电影”，与人工智能或“AI”-这种软件由复杂的交互代码层组成，类似于大脑中的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执行特定的数学运算，并将输出传递给下一层的神经细胞。在这样做的过程中，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获得的相对嘈杂的图像数据非常有用，可以告诉我们不同分子的位置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如何在细胞中发挥作用。这将是重要的信息，因为许多所谓的“超级细菌”正在出现，它们不再对抗生素产生反应，因此这可能有助于其他研究人员设计出更好的新型抗生素。正如我们在这里所做的那样，跟踪单分子并建立更好的技术将使任何参与任何细胞或有机体中DNA复制的科学家能够改进他们的工作。

项目成果

期刊论文数量（9）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

A Next Generation of Advances in Chromosome Architecture.

染色体结构的下一代进展。

DOI：
10.1007/978-1-0716-2221-6_1
发表时间：
2022
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
0
作者：
Leake MC
通讯作者：
Leake MC

Elucidating the Role of Topological Constraint on the Structure of Overstretched DNA Using Fluorescence Polarization Microscopy.

DOI：
10.1021/acs.jpcb.1c02708
发表时间：
2021-08-05
期刊：
The journal of physical chemistry. B
影响因子：
0
作者：
Backer AS;King GA;Biebricher AS;Shepherd JW;Noy A;Leake MC;Heller I;Wuite GJL;Peterman EJG
通讯作者：
Peterman EJG

Sensitive bacterial V m sensors revealed the excitability of bacterial V m and its role in antibiotic tolerance

敏感的细菌 V m 传感器揭示了细菌 V m 的兴奋性及其在抗生素耐受性中的作用