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

实时单分子方法了解 DNA 修复蛋白如何定位和消除损伤

• 批准号: BB/I003460/1
• 负责人: Neil Kad
• 金额: $ 50.09万
• 依托单位: University of Essex
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2011
• 资助国家: 英国
• 起止时间: 2011 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FI003460%2F1
• 关键词: real; time; single; molecule; approach

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.

从微生物到人DNA修复对于生命的持续至关重要。人体中的每个细胞每天都会积聚10000多个DNA损伤部位，因此有效而快速的修复至关重要。 DNA修复的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；每个人都按顺序执行一项小任务，最终导致病变修复。迄今为止，我们还不完全了解这些蛋白质如何找到损害或作为功能单位聚集在一起的物理基础。在这个项目中，我们旨在遵循模型细菌系统中核苷酸切除修复（NER）的过程。这个简单的系统涉及仅三种专用酶的相互作用，而不是人类的三十个相互作用。我们将在成像中使用最新的进步，例如快速敏感的相机，明亮的荧光标签和强大的计算机，以直接可视化这些蛋白质机器如何以及何时运行这些蛋白质。下面将更详细地讨论这一点。我们的研究提供了有关蛋白质如何在DNA上找到目标，形成复合物并允许直接可视化蛋白质级联基础机械序列的目标的重要见解。可以预料，这项研究将通过引入可用于研究其他许多过程的新技术来使其他科学家受益，并可能影响新的抗细菌药物的设计。为了研究DNA修复，我们一次可视化过程一个分子。通常，将系统研究为由数千十亿蛋白质分子组成的“合奏”。通过可视化单分子，我们能够更准确地提取有关所研究过程的顺序和时机的信息。为了使单个分子成为可能，我们将荧光信标连接到我们的蛋白质上。然后，可以使用基于最新显微镜的成像技术遵循这些标记的蛋白质。但是，要遵循蛋白质，需要考虑一个更重要的方面。当DNA可视化时，它不是一个长长的纤维，而是将DNA捆绑在一起，从而无法遵循单个标记蛋白的行为。为了克服这一点，我们开发了一种独特的方法：我们将附着在显微镜载玻片上的大珠之间的DNA暂停，以创建“ DNA Wightropes”。这些绳索使我们能够引入标记的蛋白质，并观察它们在DNA上的表现。由于维修系统使用多个蛋白质机进行工作，因此我们用不同的颜色标记了蛋白质以区分它们。 DNA修复蛋白面临着巨大的“针头”中的巨大“针头”挑战，即在一个未损坏的DNA中找到一个损坏部位（数百万至一个）。使用我们的绳索技术，我们将观察他们如何做到这一点，同时进行精确的测量，以为我们提供对这一过程的物理理解。蛋白质会沿着DNA滑动吗？在其他地方分离和重新搭架？还是两者？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复杂的问题？蜂窝能货币的角色ATP发挥了什么作用？我们还将损坏串起的DNA钢结线，并将一个量子点信标连接到损坏部位，从而为我们提供其位置。然后，我们将一起引入所有三种蛋白质，并实时观察它们如何一起修复DNA。在此提案中，我们提供了大量数据，以证明上述方法的成功，该方法使用了该领域前缘的技术，并且是我们实验室独有的。我们在这里开发的系统将为DNA维修提供新的见解，并提供使技术提供新的方法来理解其他蛋白质系统与DNA相互作用的方法。

项目成果

Understanding the coupling between DNA damage detection and UvrA's ATPase using bulk and single molecule kinetics.

• DOI: 10.1096/fj.201800899r
• 发表时间: 2019-01
• 期刊: FASEB journal : official publication of the Federation of American Societies for Experimental Biology
• 作者: Barnett JT;Kad NM
• 通讯作者: Kad NM

Using fluorescent myosin to directly visualize cooperative activation of thin filaments.

• DOI: 10.1074/jbc.m114.609743
• 发表时间: 2015-01-23
• 期刊: The Journal of biological chemistry
• 作者: Desai R;Geeves MA;Kad NM
• 通讯作者: Kad NM

Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes.

• DOI: 10.1093/nar/gkt177
• 发表时间: 2013-05
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Hughes CD;Wang H;Ghodke H;Simons M;Towheed A;Peng Y;Van Houten B;Kad NM
• 通讯作者: Kad NM

Single molecule techniques in DNA repair: a primer.

• DOI: 10.1016/j.dnarep.2014.02.003
• 发表时间: 2014-08
• 期刊: DNA REPAIR
• 影响因子: 3.8
• 作者: Hughes, Craig D.;Simons, Michelle;Mackenzie, Cassidy E.;Van Houten, Bennett;Kad, Neil M.
• 通讯作者: Kad, Neil M.

The TFIIH subunits p44/p62 act as a damage sensor during nucleotide excision repair.

• DOI: 10.1093/nar/gkaa973
• 发表时间: 2020-12-16
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Barnett JT;Kuper J;Koelmel W;Kisker C;Kad NM
• 通讯作者: Kad NM