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:
  • 金额:
    $ 50.09万
  • 依托单位:
  • 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

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。人体内的每个细胞每天都会积累超过10000个DNA损伤位点,因此有效和快速的修复至关重要。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;每一个蛋白质都在一个序列中执行一个小任务,最终导致损伤修复。到目前为止,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,而不是人体中多达30种。我们将使用成像技术的最新进展,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如何以及何时运行;这将在下面更详细地讨论。我们的研究为蛋白质如何在DNA上找到目标,形成复合物提供了重要的见解,并允许直接可视化蛋白质级联反应的机制序列。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“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连接到我们的蛋白质上。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,为了跟踪蛋白质,需要考虑一个更重要的方面。当DNA被可视化时,它不是一根长长的纤维,而是DNA被捆绑在一起,使得它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,以创建“DNA tightropes”。这些tightropes允许我们引入标记的蛋白质,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”的挑战,即在大量过量(百万分之一)的未受损DNA中找到一个损伤位点。使用我们的钢丝技术,我们将观察他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。蛋白质会沿着DNA滑动吗?分离然后在其他地方重新连接?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扮演着什么样的角色?我们也会破坏DNA的钢索,并在受损的地方安装量子点信标,从而为我们提供它的位置。然后,我们将把这三种蛋白质一起引入,在真实的时间里,我们将直接观察它们如何一起修复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有多少其他蛋白质系统与DNA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)
Using fluorescent myosin to directly visualize cooperative activation of thin filaments.
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:
  • 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:
  • 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.
Dynamics of lesion processing by bacterial nucleotide excision repair proteins.
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Neil Kad其他文献

A Branched Kinetic Pathway Facilitates Myosin Va Processivity
  • DOI:
    10.1016/j.bpj.2008.12.2959
  • 发表时间:
    2009-02-01
  • 期刊:
  • 影响因子:
  • 作者:
    Chong Zhang;Neil Kad;David M. Warshaw
  • 通讯作者:
    David M. Warshaw
A Novel DNA Repair Mechanism for the Processing of Low-Level UV-Induced Damage in Bacteria
  • DOI:
    10.1016/j.bpj.2017.11.491
  • 发表时间:
    2018-02-02
  • 期刊:
  • 影响因子:
  • 作者:
    Luke Springall;Craig Hughes;Michelle Simons;Stavros Azinas;Bennett Van Houten;Neil Kad
  • 通讯作者:
    Neil Kad

Neil Kad的其他文献

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{{ truncateString('Neil Kad', 18)}}的其他基金

Understanding dual filament regulation in muscle using single molecule imaging in vitro and in myofibrils
使用体外单分子成像和肌原纤维了解肌肉中的双丝调节
  • 批准号:
    BB/Y001621/1
  • 财政年份:
    2024
  • 资助金额:
    $ 50.09万
  • 项目类别:
    Research Grant
A multi-user access laser tweezers, fluorescence and interference microscopy facility for understanding force at the molecular level
多用户访问激光镊子、荧光和干涉显微镜设备,用于了解分子水平的力
  • 批准号:
    BB/T017767/1
  • 财政年份:
    2020
  • 资助金额:
    $ 50.09万
  • 项目类别:
    Research Grant
A Generalised Approach to Derive Functionally Active Peptide Inhibitors of Transcription Factor Activity
衍生转录因子活性的功能活性肽抑制剂的通用方法
  • 批准号:
    BB/R017921/1
  • 财政年份:
    2018
  • 资助金额:
    $ 50.09万
  • 项目类别:
    Research Grant
Reconstitution of nucleotide excision repair at the single molecule level in vitro and in vivo
体外和体内单分子水平的核苷酸切除修复重建
  • 批准号:
    BB/P00847X/1
  • 财政年份:
    2017
  • 资助金额:
    $ 50.09万
  • 项目类别:
    Research Grant
Developing and validating a new tool for simultaneous multi-channel wide-field imaging
开发并验证同步多通道宽视场成像的新工具
  • 批准号:
    BB/M019144/1
  • 财政年份:
    2015
  • 资助金额:
    $ 50.09万
  • 项目类别:
    Research Grant
Developing a novel single molecule imaging technology for application across disciplines
开发一种跨学科应用的新型单分子成像技术
  • 批准号:
    BB/M01343X/1
  • 财政年份:
    2014
  • 资助金额:
    $ 50.09万
  • 项目类别:
    Research Grant

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