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

从几年前开始，在检测非常低水平的光方面取得了巨大的技术发展，使得检测、跟踪和操纵单个生物分子成为可能。诀窍是在生物分子中加入一个标记，另一个分子在被激光等光源激发时会发出荧光。虽然我们无法看到生物分子本身，但我们可以通过检测附着在其上的标记物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分子，并利用这一特性来研究分子如何相互作用以及相互作用的时间。此外，我们可以用不同的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。这使得单分子荧光成为阐明分子机制的一种特别强大的技术：它们做什么，它们如何单独工作，它们如何一起工作，最后，它们如何在活细胞内工作。我们想应用这项技术来研究DNA修复机制。DNA修复是一项非常重要的任务，细胞为此投入了大量的能量，因为突变的DNA或错误的DNA结构如果被复制和繁殖，可能会对生物体造成严重损害。我们已经研究了一些参与这种修复机制的蛋白质，特别是结构特异性核酸内切酶，如XPF和FEN 1，它们识别异常的DNA结构并切割突出在双螺旋外的DNA链。这些蛋白质来源于古生物，这是一组非常有用的微生物模型，因为它们的DNA加工途径与高等生物（酵母，蠕虫和人类）的DNA加工途径非常相似，但更简单。我们知道这些蛋白质的结构，我们已经通过常规技术对它们进行了表征，你可以同时观察数百万个拷贝。然而，为了进一步加深对这些机制的理解，我们需要提取信息，这些信息只能通过一次观察一个分子来获得，它们相互作用的顺序，它们与受损DNA保持连接的时间以及它们如何识别异常DNA结构。

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.