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

基本信息

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

项目摘要

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.

从微生物到人类，DNA 修复对于生命的延续至关重要。人体的每个细胞每天都会积累超过 10000 个 DNA 损伤位点，因此高效、快速的修复至关重要。 DNA 修复缺陷会导致细胞死亡或持续增殖，从而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发现损伤或作为功能单元聚集在一起的物理基础。在这个项目中，我们的目标是追踪模型细菌系统中核苷酸切除修复（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，而不是人类多达三十种。我们将利用成像技术的最新进展，例如快速灵敏的相机、明亮的荧光标签和功能强大的计算机，来直接可视化这些蛋白质机器如何以及何时运行；下文对此进行了更详细的讨论。我们的研究提供了关于蛋白质如何在 DNA 上找到目标、形成复合物并允许直接可视化蛋白质级联背后的机械序列的重要见解。预计这项研究将通过引入新技术使其他科学家受益，这些新技术可用于研究许多其他过程，也可能影响新抗菌药物的设计。为了研究 DNA 修复，我们一次可视化一个分子的过程。通常，系统被研究为由数千亿个蛋白质分子组成的“整体”。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基于显微镜的成像技术来追踪这些标记的蛋白质。然而，为了追踪蛋白质，还需要考虑一个更重要的方面。当 DNA 被可视化时，它并不是一条长长的延伸纤维，而是 DNA 被捆绑在一起，因此不可能跟踪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 走钢丝”。这些走钢丝使我们能够引入标记蛋白质并观察它们在 DNA 上的行为。由于修复系统使用多个蛋白质机器来执行其工作，因此我们用不同的颜色标记蛋白质以区分它们。 DNA 修复蛋白面临着巨大的“大海捞针”挑战，即在大量（数百万比一）未损坏的 DNA 中找到一个损坏位点。利用我们的走钢丝技术，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，以便我们对这一过程有一个物理上的了解。蛋白质会沿着 DNA 滑动吗？在其他地方分离并重新连接？或者两者都有？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合物？细胞能量货币 ATP 扮演什么角色？我们还将损坏悬挂的 DNA 钢丝，并将量子点信标连接到损坏位置，从而为我们提供其位置。然后我们将同时引入所有三种蛋白质，并实时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方法的成功，该方法使用了该领域的前沿技术，并且是我们实验室独有的。我们在这里开发的系统将为 DNA 修复提供新的见解，并提供使能技术，以提供一种新的方式来了解许多其他蛋白质系统与 DNA 的相互作用。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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
影响因子：
0
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
影响因子：
0
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.