Single-molecule analysis of double-stranded DNA break repair in living bacteria

我们的研究使用超灵敏的显微镜来观察DNA修复过程中发生的重要过程，DNA是主要以染色体形式存储遗传信息的分子。DNA修复是活细胞修复其遗传物质不同类型损伤的不同方式的总称。因此，DNA修复对于所有生物体的生存和生长至关重要。具体来说，我们的工作重点是修复断裂的染色体的过程中，当他们暴露于DNA双螺旋的两条链上的断裂;这种断裂被称为“双链断裂”。在细菌中，这些DNA断裂通常由两种称为RecBCD和RecA的蛋白质机器固定，它们以协调的方式发挥作用。RecBCD机器设法在细胞中大量完整的DNA中找到断裂的DNA末端，并去除部分断裂的DNA末端，留下特殊的DNA结构。这种结构然后被RecA机器的许多副本识别，该机器形成坚硬的DNA细丝，开始了对染色体完整副本的迷人而神秘的搜索。这个过程需要快速和特异性，因为任何错误没有及时修复都可能导致危险的突变甚至细胞死亡。我们对DNA断裂如何修复的了解，大部分来自对试管中纯化蛋白质和DNA的研究;这些研究涉及RecBCD和RecA机器与DNA序列和辅助蛋白的简单混合，这些蛋白质加速或促进了这一过程。然而，由于细胞中存在无数其他生物成分，以及由于基因包装在“细菌类核”中的方式，实际生物体中的DNA修复机制可能非常不同，“细菌类核”是由细菌DNA及其一些蛋白质组成的紧密包装结构。为了提供一个活细胞中DNA修复复杂性的例子，我们可以考虑RecA的任务：在整个染色体中搜索50个DNA字母，这是一个长度超过5，000，000个字母并且高度折叠的分子。另一个复杂性的例子与其他蛋白质有关，这些蛋白质可以干扰或促进RecBCD的功能，当RecBCD试图固定通过用抗生素治疗病原菌而形成的DNA末端时。为了研究DNA在活细胞的自然环境中的修复过程，我们将使用先进的荧光显微镜来观察RecBCD机器如何搜索，发现，并在活细菌细胞内的伴侣RecA的帮助下帮助断裂的DNA结束。我们将使用细菌大肠杆菌，这是一个简单的模式生物了解生物机制。我们工作的一个特点是使用特殊的显微镜（“单分子荧光显微镜”）进行。这种显微镜经过精心设计，可以检测和监测活细胞内的单个荧光分子（与需要数千或数百万个荧光分子的传统显微镜相反）。使用我们强大的显微镜记录电影并跟踪蛋白质移动时的位置，我们将看到蛋白质如何搜索并找到不同类型的断裂DNA末端，无论是在染色体上，还是在合成类型的断裂DNA上，使我们能够更直接地看到这个过程，并在真实的时间。我们还将研究单个RecBCD机器如何切割DNA末端，为RecA机器的着陆做准备。最后，我们将研究RecA如何形成能够搜索DNA的正确拷贝以修复受损部分的DNA类型。我们的研究将提高我们对DNA修复如何在活细胞中工作的理解，并帮助其他科学家研究其他生物体（如人类）的DNA修复，以及开发新的药物，通过禁用危险微生物的DNA修复机制来改善人类，动物和植物的健康。

Our study uses ultra-sensitive microscopes to observe important processes taking place during the repair of DNA, the molecule wherein the genetic information is stored mainly in the form of chromosomes. DNA repair is the general term for the collection of the different ways living cells fix different types of damage to their genetic material. As such, DNA repair is central for the survival and growth of all living organisms. Specifically, our work focuses on the process of repairing broken chromosomes when they are exposed to breaks on both strands of the DNA double helix; such breaks are known as "double-stranded breaks". In bacteria, these DNA breaks are often fixed by two protein machines called RecBCD and RecA, that act in a well coordinated fashion. The RecBCD machine manages to find the broken DNA ends amongst huge amounts of intact DNA in the cell and removes part of the broken DNA end to leave a special DNA structure. This structure is then recognised by many copies of the RecA machine that forms of stiff DNA filament that embarks on a fascinating and mysterious search for an intact copy of the chromosome. This process needs to be fast and specific, since any errors not fixed in time can lead to dangerous mutations or even cell death.Much of what we know about how DNA breaks are fixed comes from studies with purified proteins and DNA in the test tube; these involve simple mixtures of the RecBCD and RecA machines with DNA sequences and helper proteins that accelerate or facilitate the process. However, the mechanisms of DNA repair in actual living organisms can be very different, due to the myriad of other biological components that are present in cells, and due to the way that the genes are packaged in the "bacterial nucleoid", which is a tightly packed structure made of the bacterial DNA and some of its proteins. To provide an example of the complexity that characterises DNA repair in living cells, one can consider the task of RecA: to search just 50 letters of DNA within the entire chromosome, which is a molecule longer than 5,000,000 letters and highly folded. Another example of the complexity has to do with other proteins that can interfere with or facilitate the function of RecBCD when it tries to fix DNA ends that have been formed by treating pathogenic bacteria with antibiotics.To study the process of DNA repair in its natural environment of living cells, we will use advanced fluorescence microscopy to look at how the RecBCD machine searches, finds, and helps broken DNA ends with the help of its partner RecA inside living bacterial cells. We will use the bacterium Escherichia coli, which is a simple model organism for understanding biological mechanisms. A special feature of our work is that it is performed using a special microscope (a "single-molecule fluorescence microscope"). This microscope is carefully designed to allow detection and monitoring of individual (single) fluorescent molecules inside living cells (as opposed to conventional microscopes that require thousands or millions of fluorescent molecules).Using our powerful microscope to record movies and track the position of the proteins as they move, we will see how proteins search and find different types of broken DNA ends, either on the chromosome or on synthetic types of broken DNA that allow us to see the process more directly and in real time. We will also study how individual RecBCD machines cut out the DNA end to prepare the ground for the landing of the RecA machines. Finally, we will study how RecA forms the type of DNA that is able to search for the correct copy of the DNA to fix the damaged part. Our studies will improve our understanding of how DNA repair works in living cells, and help other scientists to study DNA repair in other organisms (such as humans), as well as to develop new pharmaceuticals that will improve the health of humans, animals and plants by disabling the DNA repair machinery of dangerous microbes.