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
• 负责人: Neil Kad
• 金额: $ 41.51万
• 依托单位: University of Kent
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FP00847X%2F1
• 关键词: Reconstitution; nucleotide; excision; repair; single

Simply stepping outside on a sunny day exposes the skin to enough ultraviolet radiation (UV) to cause blistering and the formation of cancerous tumours. Why this doesn't occur is due to enzymes present in every cell that scan DNA for damage and then initiate repair. Xeroderma pigmentosum (XP) is one of a number of diseases caused by deficiencies in this repair pathway and for individuals with XP this leads to skin blistering, cancer and neurological dysfunction. A complete lack of these nucleotide excision repair (NER) enzymes is lethal. Because most organisms are exposed to UV, this mechanism of DNA repair is conserved across all forms of life. In humans over 30 enzymes are involved in NER, whereas in bacteria only 6 enzymes are required. Therefore understanding NER at the simpler bacterial level will provide insight into the human equivalent. Despite decades of research into NER there is surprisingly little known about the precise details. The components are well-established but how they work together is still uncertain. The main aim of our work is to understand how the bacterial system works as a whole, but still at the molecular level. This is important because the classical approach of studying individual components may miss the formation of enzyme complexes or overstate the importance of individual components. This is very complex and therefore we study single molecules to simplify the system. We aim to directly watch complexes forming, their mechanisms of damage location and the recruitment of other components. These are all physical concepts; a protein has to search through a sea of undamaged DNA to find the lesion, somehow it must communicate with other proteins to signal that it has achieved this goal and then organise these other proteins onto the site of damage. Only through single molecule imaging of a complex mixture of components can we get a true picture of how DNA is repaired. To take this further we are also proposing to image these processes in live bacteria. We will use cutting edge techniques to isolate single molecules within cells and study how they behave alone and with each other. This is immensely exciting; the prospect of visualising single molecule processes in their native environments is a very new field of study. These combined approaches will offer a complete view of how DNA repair occurs in vitro and in vivo.Not only will this project improve our understanding of bacteria repair it will serve as a proxy for understanding how proteins interact with DNA more generally. There is a gap in our toolset from cell biology to single molecule imaging that we will fill during this project. Therefore the tools and techniques that we develop will find application across a wide range of problems. Ultimately, the knowledge gained from this study will inform studies of human equivalent systems, such as XP. This will have considerable impact on the lives of individuals with this highly debilitating condition.

只需在晴天踏出外面，就会使皮肤暴露于足够的紫外线辐射（UV），以引起起泡和癌性肿瘤的形成。为什么不发生这种情况是由于每个细胞中存在扫描DNA的酶以进行损坏然后启动修复。心虫色素（XP）是该修复途径缺陷引起的多种疾病之一，对于XP的个体，这会导致皮肤发泡，癌症和神经功能障碍。这些核苷酸切除修复（NER）酶完全缺乏致命。由于大多数生物都暴露于紫外线，因此这种DNA修复机制在各种形式的生命中都保存下来。在人类中，超过30个酶参与了NER，而在细菌中，仅需要6种酶。因此，在简单的细菌水平上了解NER将提供对人类当量的见解。尽管对NER进行了数十年的研究，但令人惊讶的是，关于确切细节鲜为人知。这些组件已经建立了良好，但是它们如何一起工作仍然不确定。我们工作的主要目的是了解细菌系统的整体作用，但仍处于分子水平。这很重要，因为研究各个组件的经典方法可能会错过酶复合物的形成或夸大了单个成分的重要性。这很复杂，因此我们研究单分子以简化系统。我们的目标是直接观察形成的复合物，它们的损害位置机制以及其他组件的募集。这些都是物理概念；一种蛋白质必须在未损坏的DNA海洋中搜索以找到病变，以某种方式必须与其他蛋白质进行通信，以表明它已经实现了该目标，然后将这些其他蛋白质组织到损伤部位。只有通过组件的复杂混合物的单分子成像，我们才能真正了解如何修复DNA。为了进一步，我们还建议在活细菌中对这些过程进行图像。我们将使用尖端技术来分离细胞内的单个分子，并研究它们单独和彼此之间的行为。这非常令人兴奋。在本机环境中可视化单分子过程的前景是一个非常新的研究领域。这些合并的方法将提供有关DNA修复方式在体外和体内如何进行的完整视图。仅此项目将不仅可以提高我们对细菌修复的理解，还可以作为理解蛋白质如何更普遍地与DNA相互作用的代理。从细胞生物学到单分子成像，我们的工具集存在差距，我们将在此项目中填充。因此，我们开发的工具和技术将在各种问题上找到应用。最终，从这项研究中获得的知识将为人类当量系统（例如XP）的研究提供信息。这将对患有这种高度衰弱的状况的个人的生活产生相当大的影响。

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

Combining cancer chemotherapeutics with bacterial DNA repair inhibitors to develop novel antimicrobials

• DOI: 10.1101/2023.03.17.532951
• 发表时间: 2023-03
• 期刊: bioRxiv
• 作者: L. Bernacchia;Arya Gupta;A. Paris;Alexandra A. Moores;N. Kad

Recruitment of UvrBC complexes to UV-induced damage in the absence of UvrA increases cell survival.

• DOI: 10.1093/nar/gkx1244
• 发表时间: 2018-02-16
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Springall L;Hughes CD;Simons M;Azinas S;Van Houten B;Kad NM
• 通讯作者: Kad NM

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

Identification of the target and mode of action for the prokaryotic nucleotide excision repair inhibitor ATBC.

• DOI: 10.1042/bsr20220403
• 发表时间: 2022-06-30
• 期刊: Bioscience reports
• 影响因子: 4