Reconstitution of nucleotide excision repair at the single molecule level in vitro and in vivo

在阳光明媚的日子里，只要走到户外，皮肤就会暴露在足够的紫外线辐射（UV）下，导致起泡和癌性肿瘤的形成。为什么这不会发生是由于酶存在于每个细胞中，扫描DNA的损伤，然后启动修复。着色性干皮病（XP）是由这种修复途径的缺陷引起的许多疾病之一，对于患有XP的个体，这会导致皮肤起泡、癌症和神经功能障碍。完全缺乏这些核苷酸切除修复（NER）酶是致命的。由于大多数生物体都暴露在紫外线下，这种DNA修复机制在所有形式的生命中都存在。在人类中，有超过30种酶参与NER，而在细菌中只需要6种酶。因此，在更简单的细菌水平上理解NER将提供对人类等同物的深入了解。尽管对NER进行了数十年的研究，但令人惊讶的是，对确切的细节知之甚少。这些组成部分已经很好地建立起来，但它们如何协同工作仍然不确定。我们工作的主要目的是了解细菌系统作为一个整体如何工作，但仍然是在分子水平上。这一点很重要，因为研究单个组分的经典方法可能会错过酶复合物的形成或夸大单个组分的重要性。这是非常复杂的，因此我们研究单个分子来简化系统。我们的目标是直接观察复合物的形成，它们的损伤定位机制和其他成分的招募。这些都是物理概念;蛋白质必须在一片未受损的DNA海洋中搜索以找到病变，它必须以某种方式与其他蛋白质通信以发出它已经实现这一目标的信号，然后将这些其他蛋白质组织到损伤部位。只有通过对复杂的成分混合物进行单分子成像，我们才能真正了解DNA是如何修复的。为了更进一步，我们还提议在活细菌中对这些过程进行成像。我们将使用尖端技术来分离细胞内的单个分子，并研究它们如何单独和相互作用。这是非常令人兴奋的;在其自然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。这些结合的方法将提供DNA修复如何在体外和体内发生的完整视图。该项目不仅将提高我们对细菌修复的理解，还将作为理解蛋白质如何与DNA更普遍地相互作用的代理。在我们的工具集中，从细胞生物学到单分子成像，我们将在这个项目中填补空白。因此，我们开发的工具和技术将在广泛的问题中找到应用。最终，从这项研究中获得的知识将为人类等效系统（如XP）的研究提供信息。这将对患有这种高度衰弱疾病的个人的生活产生相当大的影响。

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.