Organization and function of structure-specific endonucleases: single-molecule studies of fluorescently labelled NER complexes

结构特异性核酸内切酶的组织和功能：荧光标记 NER 复合物的单分子研究

基本信息

批准号：
BB/E014674/1
负责人：
Carlos Penedo
金额：
$ 42.7万
依托单位：
University of St Andrews
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2007
资助国家：
英国
起止时间：
2007 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FE014674%2F1
关键词：
Organization function structure specific endonucleases

项目摘要

Since a few years ago, tremendous technical developments in the detection of very low levels of light have made it possible to detect, track, and manipulate single biomolecules. The trick is to incorporate into the biomolecule a label, another molecule that emits fluorescence when excited by a light source like a laser. Although we can not see the biomolecule itself, we can identify its position by detecting the fluorescence signature coming from the label attached to it. Thus, we can differentiate molecules by labelling them with different fluorescence colours and use this property to investigate how molecules interact and for how long. Also, we can label different parts of the same molecule with different colours to get information about their relative movement. This has made single-molecule fluorescence a particularly powerful technique in elucidating mechanisms of molecular machineries: what they do, how they work individually, how they work together, and finally, how they work inside live cells. We want to apply this technique to study the DNA repair machinery. DNA repair is a very important task and cells devote a lot of energy to this, as mutated DNA or wrong DNA structures can cause severe damage in living organisms if they are copied and propagate. We have studied some of the proteins that participate in this repair mechanism, in particular structure-specific endonucleases such as XPF and FEN1 that recognize anomalous DNA structures and cut DNA strands protruding outside the double helix. These proteins are derived from archaeal organisms, a group of microbes that are very useful models because their DNA processing pathways are rather similar but simpler than those in higher organisms (yeast, worms and humans). We know the structure of these proteins and we have characterized them by conventional techniques, where you look at millions of copies at the same time. However, to further advance in our understanding of these mechanisms we need to extract the information that is only accessible by looking one molecule at a time, in which order they interact, for how long they remain attached to the damage DNA and how they recognize the anomalous DNA structures.

从几年前开始，检测到非常低的光的巨大技术发展使得可以检测，跟踪和操纵单一生物分子。诀窍是将其掺入标签中的生物分子中，这是另一个由像激光这样的光源激发时发出荧光的分子。尽管我们看不到生物分子本身，但我们可以通过检测来自附着的标签的荧光特征来识别其位置。因此，我们可以通过用不同的荧光颜色标记分子来区分它们，并使用该特性来研究分子的相互作用和时间的时间。另外，我们可以用不同颜色标记同一分子的不同部分，以获取有关其相对运动的信息。这使单分子荧光成为阐明分子机制机制的特别强大的技术：他们的工作，他们如何单独工作，如何一起工作，最后是它们在活细胞内的工作方式。我们想应用此技术来研究DNA维修机械。 DNA修复是一项非常重要的任务，并且细胞为此投入了大量能量，因为如果复制和繁殖，突变的DNA或错误的DNA结构可能会在生物体中造成严重损害。我们研究了一些参与该修复机制的蛋白质，特别是结构特异性的核酸内切酶，例如XPF和FEN1，它们识别异常的DNA结构并切断了双螺旋外突出的DNA链。这些蛋白质来自古细菌，这是一组非常有用的模型的微生物，因为它们的DNA加工途径相当相似，但比较高生物体（酵母，蠕虫和人）更简单。我们知道这些蛋白质的结构，并且通过传统技术来表征它们，您可以同时查看数百万份。但是，为了进一步促进我们对这些机制的理解，我们需要提取只能通过一次查看一个分子（在其相互作用的顺序）上可以访问的信息，以使它们与损伤DNA保持依恋时间以及它们如何识别异常的DNA结构。