A real-time single molecule approach to understand how DNA repair proteins locate and remove damage

从微生物到人类，DNA修复对生命的延续至关重要。人体每个细胞每天都会累积超过10000个DNA损伤位点，因此高效、快速的修复是必不可少的。DNA修复缺陷导致细胞死亡或持续增殖，分别导致过早衰老或肿瘤形成。修复是由蛋白质介导的；每一个都按顺序执行一个小任务，最终导致损伤修复。到目前为止，我们还没有完全了解这些蛋白质如何发现损伤或作为功能单位聚集在一起的物理基础。在这个项目中，我们的目标是在一个模型细菌系统中跟踪核苷酸切除修复（NER）的过程。这个更简单的系统只涉及三种专用酶的相互作用，而不是人类的30种。我们将利用成像方面的最新进展，如快速灵敏的相机、明亮的荧光标签和强大的计算机，直接可视化这些蛋白质机器的运作方式和时间；下面将对此进行更详细的讨论。我们的研究为蛋白质如何在DNA上找到它们的靶标，形成复合物，并允许直接可视化蛋白质级联的机制序列提供了重要的见解。预计这项研究将通过引入可用于研究许多其他过程的新技术，并可能影响新的抗菌药物的设计，从而使其他科学家受益。为了研究DNA修复，我们一次观察一个分子的修复过程。通常，系统被研究为由数万亿个蛋白质分子组成的“集合体”。通过可视化单个分子，我们能够更准确地提取有关所研究过程的顺序和时间的信息。为了能够看到单个分子，我们将称为量子点的荧光信标附着在蛋白质上。然后可以使用最先进的显微镜成像技术跟踪这些标记的蛋白质。然而，为了遵循蛋白质，需要考虑一个更重要的方面。当DNA被可视化时，它不是一个长长的伸展的纤维，相反，DNA是捆绑在一起的，这使得不可能遵循单个标记蛋白质的行为。为了克服这个问题，我们开发了一种独特的方法：我们将DNA悬浮在附着在显微镜载玻片上的大珠子之间，形成“DNA绳索”。这些钢丝让我们可以引入标记蛋白，观察它们在DNA上的表现。由于修复系统使用多台蛋白质机器来进行工作，我们用不同的颜色标记蛋白质来区分它们。DNA修复蛋白面临着巨大的“大海捞针”的挑战，要在大量（百万分之一）未受损的DNA中找到一个损伤位点。使用我们的绳索技术，我们将观察它们是如何做到这一点的，同时进行精确的测量，为我们提供对这一过程的物理理解。蛋白质沿着DNA滑动吗？在其他地方分离并重新连接？还是两个?我们还将能够解决该领域长期存在的问题，例如有多少蛋白质形成一个复合体？细胞能量货币ATP起什么作用？我们还将破坏DNA绳索，并将量子点信标连接到受损部位，从而为我们提供其位置。然后，我们将把这三种蛋白质一起引入，实时地，我们将直接观察它们如何共同修复DNA。在本提案中，我们提供了大量数据来证明上述概述方法的成功，该方法使用的技术处于该领域的前沿，并且是我们实验室独有的。我们在这里开发的系统将为DNA修复提供新的见解，并提供使能技术，为了解许多其他蛋白质系统如何与DNA相互作用提供新的方法。

From microbes to man DNA repair is crucial to the continuance of life. Each cell in the human body accumulates over 10000 sites of DNA damage every day, therefore efficient and rapid repair is essential. Defects in DNA repair result in cell death or continual proliferation, leading to premature ageing or tumour formation respectively. Repair is mediated by proteins; each one performs a small task in a sequence that eventually leads to lesion repair. To date we do not fully understand the physical basis of how these proteins find damage or come together as functional units. In this project we aim to follow the process of nucleotide excision repair (NER) in a model bacterial system. This simpler system involves the interplay of just three dedicated enzymes instead of up to thirty in humans. We will use recent advances in imaging such as fast sensitive cameras, bright fluorescent tags and powerful computers to directly visualise how and when these protein machines operate; this is discussed in more detail below. Our research offers important insights into how proteins find their targets on DNA, form complexes and permits direct visualisation of the mechanistic sequence underlying a protein cascade. It is anticipated that this research will benefit other scientists by introducing new techniques that could be used to investigate a number of other processes and may also impact the design of new anti-bacterial drugs. To study DNA repair we visualise the process one molecule at a time. Normally, systems are studied as 'ensembles' consisting of thousands of billions of protein molecules. By visualising single molecules we are able to extract information much more accurately about both the order and timing of the process being studied. To make it possible to see a single molecule we attach fluorescent beacons called a quantum dots to our proteins. These tagged proteins can then be followed using a state-of-the-art microscope based imaging technique. However to follow the proteins one more important aspect needs to be considered. When DNA is visualised it is not a long stretched out fibre, instead DNA is bundled, making it impossible to follow the behaviour of a single tagged protein. To overcome this we have developed a unique approach: we suspend the DNA between large beads attached to a microscope slide to create 'DNA tightropes'. These tightropes allow us to introduce tagged proteins and watch how they behave on DNA. Since the repair system uses multiple protein machines to carry out its work, we have tagged the proteins with different colours to distinguish them. DNA repair proteins face the enormous 'needle in a haystack' challenge of finding one damage site amongst a vast excess (millions to one) of undamaged DNA. Using our tightrope technology we will watch how they do this, and at the same time make precise measurements to provide us with a physical understanding of this process. Do the proteins slide along the DNA? Detach and reattach elsewhere? Or both? We will also be able to address long held questions in the field such as how many proteins form a complex? And what role ATP, the cellular energy currency, plays? We will also damage the strung up DNA tightropes and attach a quantum dot beacon to the damage site thus providing us with its location. Then we will introduce all three proteins together and, in real time, we will directly observe how they work together to repair the DNA. In this proposal we present a large amount of data to demonstrate the success of the above outlined approach, which uses technology that is at the leading edge of the field and is unique to our laboratory. The system we are developing here will offer a new insight into DNA repair and also provide enabling technology to offer a new way of understanding how many other protein systems interact with DNA.